Dual function antibodies specific to glycosylated pd-l1 and methods of use thereof

ABSTRACT

Antibodies that bind specifically to glycosylated PD-L1 relative to unglycosylated PD-L1, block binding of PD-L1 to PD-1 and promote internalization and degradation of PD-L1 are provided. Antibodies that recognize specific epitopes on glycosylated PD-L1 protein and that exhibit the dual functions of both blocking the binding of PD-L1 to PD-1 and also facilitating the internalization of PD-L1 on cells are provided. In some aspects, PD-L1 polypeptides comprising glycosylated amino acid residues at amino and carboxy terminal positions of the PD-L1 extracellular domain are also provided. Methods for using such antibodies for the treatment of cancer, particularly PD-L1 positive cancer, are also provided.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 23, 2017, isnamed 24258_105007_SL.txt and is 40,452 bytes in size.

RELATED FIELDS

The present disclosure relates generally to the fields of molecularbiology, medicine and oncology. More particularly, new antibodies thatspecifically bind glycosylated PD-L1 and their use for treating cancersare provided.

BACKGROUND

Perpetuation of T-cell activation has drastically reshaped the treatmentof a broad spectrum of malignant cancers. For instance, the developmentof ipilimumab, the first FDA approved checkpoint blockade targetingT-cell response made treating metastatic melanoma probable (Hodi, F. S.et al., 2010, NEJM, 363:711-723; Robert, C. et al., 2013, Clin. CancerRes., 19:2232-2239; and Robert, C. et al., 2011, NEJM, 364:2517-2526).While the anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) monoclonalantibody showed promising results in treating melanoma patients,second-generation checkpoint inhibitors targeting both PD-1 and PD-L1have demonstrated better clinical activity and safety in phase IIIclinical trials (Topalian, S. L. et al., 2012, NEJM, 366:2443-54; andBrahmer, J. R. et al., 2012, NEJM, 366:2455-2465). Because PD-L1 alsopossesses oncogenic potential that induces cancer cells progression(Topalian, S. L. et al., Id.; Page, D. B. et al., 2014, Ann. Rev. Med.,65:185-202). In addition to its immunosuppression activity, targetingthe PD-1/PD-L1 interaction provides dual efficacy by blockingimmunosuppression via PD-1 while reducing cell progression via PD-L1 andis expected to have more sensitive outcome (Topalian, S. L. et al., Id.;Brahmer, J. R. et al., Id.; and Hamid, O., 2013, NEJM, 369:134-144). In2014, there were over 10 clinical trials ongoing in the U.S. testing theefficacy of anti-PD-L1 and/or anti-PD-1 antibodies either as a singleagent or in combination (Page, D. B. et al., Id.). The US FDA hasapproved two anti-PD-1 therapeutic antibodies for treatment of certaincancers KEYTRUDA® (pembrolizumab) and OPDIVO® (nivolumab). However, thepathophysiological function and regulatory mechanism of PD-L1 remainsincompletely defined.

Reawakening silenced immune response, particularly effector T-cells, hasbeen recently added to a repertoire of treatment options that includesurgical removal, chemotherapy, radiotherapy, and targeted therapies.While the use of anti-CTLA-4 monoclonal antibody (Dunn, G. P., et al.,2002, Nat. Immunol., 3:991-998; and Leach, D. R., et al., 1996, Science,271:1734-1736) initially demonstrated success in treating metastaticmelanoma, it has been shown to also induce an autoimmune response.Unlike anti-CTLA-4 antibodies, which affect only immune cells,anti-PD-L1 antibodies and anti-PD-1 antibodies act at a cellular leveland at tumor sites to block the interaction between PD-1-expressingeffector T-cells and PD-L1-expressing tumor cells. This creates a dualimpact from both the tumor cell and the T-cell, thereby limiting theadverse effects and providing better therapeutic efficacy (Okazaki, T.,et al., 2013, Nature immunology, 14:1212-1218). A better understandingof the pathophysiological mechanism underlying the PD-L1 mediated immunesuppression and pro-oncogenic effect on tumor cells is needed to developmore robust therapeutics targeting this pathway. There remains a needfor new and more effective therapeutics and methodologies thatsuccessfully target the PD-1/PD-L1 pathway and activate effector cellsof the immune system to attack the tumor cells and treat cancers.

SUMMARY

The inventors have discovered that glycosylation of PD-L1 (also known asCD274, PDCD1L1, or B7-H1) expressed on tumor cells promotes or enhancesbinding to PD-1 expressed on immune effector cells, such as T cells,thereby increasing the suppression of T cell activity against the tumorcells. The inventors further discovered that glycosylation of PD-L1 canstabilize PD-L1 expression on the cell surface, thus reducing the rateof internalization and intracellular degradation of the PD-L1. Theinventors have identified antibodies that preferentially bind toglycosylated human PD-L1 polypeptide relative to unglycosylated humanPD-L1 polypeptide. As used herein, such antibodies that preferentiallybind glycosylated human PD-L1 are referred to as “anti-glycPD-L1antibodies.” The anti-glycPD-L1 antibodies as described herein also havea dual functionality in that they block the PD-L1/PD-1 interaction andalso promote internalization and intracellular degradation of PD-L1 onPD-L1-expressing cells; thus, these antibodies are termed “dualfunction” antibodies. Accordingly, such anti-glycPD-L1 antibodies thatexhibit the dual functionality of inhibiting PD-1/PD-L1 binding andpromoting PD-L1 internalization and degradation are provided.

Further provided are methods of treating cancer, particularly cancerswhose cells express or overexpress PD-L1, in a subject in need thereofby administering one or more anti-glycPD-L1 antibodies. Theanti-glycPD-L1 antibodies as described herein inhibit or block theinteraction between PD-1 and PD-L1 and also reduce the levels of PD-L1on the tumor cells, and thereby inhibit immunosuppression which resultsfrom the PD-1/PD-L1 interaction and promote the perpetuation of thecytotoxic activity of PD-1-expressing effector T-cells against tumorcells that express PD-L1. By inhibiting the PD-1/PD-L1 interaction andreducing levels of glycosylated PD-L1 on the tumor cell surface, theanti-glycPD-L1 antibodies as described can enhance effector T-cellresponses and mediate anti-tumor activity. As used herein, the termstumor and cancer are used interchangeably. Unless otherwise indicated,“PD-L1” as used herein refers to PD-L1 protein, polypeptide, or peptide,particularly human PD-L1 (the amino acid sequence of which is SEQ ID NO:1); and “PD-1” refers to PD-1 protein, polypeptide, or peptide,particularly human PD-1.

The inventors have further found that human PD-L1 is glycosylated atfour sites in the extracellular domain at amino acid positions N35,N192, N200 and/or N219 of the human PD-L1 protein, e.g., as set forth inSEQ ID NO: 1. The anti-glycPD-L1 antibodies as described may bind to oneor more of these sites and, for example, may not bind to PD-L1 that hasa mutation (for example, substitution of glutamine for asparagine withinthe glycosylation consensus sequence) at one of more of theseglycosylation sites and, thus, is not glycosylated at those one or moresites. Accordingly, in some embodiments, the anti-glycPD-L1 antibodyspecifically binds to one or more glycosylation motifs in the PD-L1glycopolypeptide or peptides thereof. In some embodiments, theanti-glycPD-L1 antibody binds to a PD-L1 glycopeptide which comprises aglycosylation motif and the adjacent peptide. In some embodiments, theanti-glycPD-L1 antibody binds to a peptide sequence that is located nearone or more of the glycosylation motifs in three dimensions.Accordingly, in embodiments, the anti-glycPD-L1 antibody recognizes andselectively binds to a conformational epitope of glycosylated PD-L1. Byway of example, in certain embodiments, the anti-glycPD-L1 antibodybinds to glycosylated PD-L1 with a K_(d) of less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, less than 50%, less than 45% of the K_(d) exhibited relativeto unglycosylated PD-L1, but in embodiments, no more than 5%, 10%, 15%,20% or 25% of the K_(d) exhibited relative to unglycosylated PD-L1, butstill exhibits the dual anti-glycosylated PD-L1 function. It is to beunderstood that values in between as well as equal to the foregoingK_(d) values are encompassed. In an embodiment the anti-glycPD-L1antibody binds to glycosylated PD-L1 with a K_(d) of less than half ofthe K_(d) exhibited relative to unglycosylated PD-L1, but still exhibitsthe dual anti-glycosylated PD-L1 function. In an embodiment, theanti-glycPD-L1 antibody binds to glycosylated PD-L1 protein with a K_(d)at least 5 times less than the K_(d) exhibited relative tounglycosylated PD-L1. In an embodiment, the anti-glycPD-L1 antibodybinds to glycosylated PD-L1 protein with a K_(d) at least 10 times lessthan the K_(d) exhibited relative to unglycosylated PD-L1 protein. Incertain embodiments, the anti-glycPD-L1 antibody preferentially binds tocells expressing the WT glycosylated PD-L1 with at least 1.5 times, 2,times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or10 times greater frequency than to cells expressing unglycosylated PD-L1as assayed in, for example, a cell flow cytometry assay in which thecells expressing WT PD-L1 and unglycosylated PD-L1 are mixed anddifferentially labeled, and then contacted with the antibody to beassayed labeled with a detectable marker, for example, as described inExample 6, and as measured, for example, by the measured fluorescenceintensity (MFI) for the two populations of cells when the antibody islabeled with a fluorescent marker. In an embodiment, the anti-glycPD-L1antibody selectively binds to glycosylated PD-L1 protein with anaffinity of from 5-20 nM, 5-10 nM, or 10-20 nM. In an embodiment, theantibody is a monoclonal antibody and, more preferably a chimeric orhumanized or human antibody. The terms “specifically bind” and“selectively bind” are used interchangeably herein.

The anti-glycPD-L1 antibodies described herein specifically bindglycosylated PD-L1 versus non-glycosylated PD-L1; reduce, block, orinhibit binding of PD-L1 to PD-1; and promote internalization andintracellular degradation of PD-L1. In certain embodiments, the dualfunction anti-glycPD-L1 antibodies bind PD-L1 epitopes that arenonlinear, conformational epitopes. In addition, and without being boundby theory, the anti-glycPD-L1 antibodies bind PD-L1 epitopes thatcontain or are proximal in the three dimensional structure of PD-L1 toglycosylated amino acids; such glycosylated regions of PD-L1 arebelieved to be particularly involved in the PD-L1/PD-1 interaction. Bybinding epitope amino acids in regions of PD-L1 that are glycosylated,the anti-glycPD-L1 antibodies as described may effectively function tomask or neutralize binding sites or regions of the glycosylated PD-L1protein that are involved in the PD-L1/PD-1 interaction and/or in thestabilization of PD-L1 expression on the tumor cell surface.

The anti-glycPD-L1 antibodies as described exert dual functionalities byvirtue of their binding to glycosylated PD-L1 and are thus termed ‘dualfunction’ antibodies herein. It is believed, although not being bound bytheory, that the N-terminal glycosylation site of the ECD (N35) isinvolved primarily with PD-L1/PD-1 binding, while the more C-terminalglycosylation sites (N192, N200 and N219) of the ECD are involvedprimarily with the stabilization of PD-L1 on cell membranes, althougheach of these regions may contribute to both PD-L1/PD-1 binding and thestabilization of PD-L1 on the membranes. Accordingly, anti-glycPD-L1antibodies that bind to, block and/or mask the glycosylation at N35 mayinhibit PD-L1/PD-1 binding and antibodies that bind to, block and/ormask the glycosylation at N192, N200 and/or N219 promote PD-L1internalization and degradation. The antibodies as provided herein haveboth functionalities.

Provided in particular aspects are dual function anti-glycPD-L1monoclonal antibodies STM073 (which has a V_(H) domain amino acidsequence of SEQ ID NO: 3 and a V_(L) domain amino acid sequence of SEQID NO: 11, as also provided in Table 3, infra) and STM108 (which has aV_(H) domain amino acid sequence of SEQ ID NO: 19 and a V_(L) domainamino acid sequence of SEQ ID NO: 27, as also provided in Table 3,infra), and binding fragments thereof specific for glycosylated PD-L1,as well as chimeric and humanized forms thereof. In one embodiment,STM073 specifically binds an epitope on PD-L1 corresponding to aminoacid residues encompassing positions H69, Y112, R113 and K124 of thehuman PD-L1 amino acid sequence of SEQ ID NO: 1 herein. This STM073epitope is non-contiguous within the amino acid sequence and is aconformational epitope. In one embodiment, the portion of the humanPD-L1 polypeptide encompassing the STM073 MAb epitope has the sequenceVHGEEDLKVQH------DAGVYRCMISYGGADYKRITV (SEQ ID NO: 85), in which theamino acid residues H69, Y112, R113 and K124, which comprise the epitoperecognized by MAb STM073, are underlined and the dashes between aminoacid residue histidine (H) at position 78 and amino acid residueaspartic acid (D) at position 108 represent amino acids at positions79-107 of the human PD-L1 amino acid sequence of SEQ ID NO: 1. Providedare antibodies that bind the STM073 epitope and antibodies that competefor binding to PD-L1 with STM073.

In another embodiment, the STM108 MAb specifically binds an epitope onPD-L1 corresponding to amino acid residues encompassing positions S80,Y81, K162 and S169 of the human PD-L1 amino acid sequence of SEQ ID NO:1 herein. This STM108 epitope is non-contiguous within the amino acidsequence and is a conformational epitope. In one embodiment, the portionof the human PD-L1 polypeptide encompassing the STM073 MAb epitope hasthe sequence LKVQHSSYRQR------EGYPKAEVIWTSSDHQ (residues 74-173 of SEQID NO: 1) in which the amino acid residues S80, Y81, K162 and S169,which comprise the epitope recognized by MAb STM108, are underlined andthe dashes between amino acid residue arginine (R) at position 84 andamino acid residue glutamic acid (E) at position 158 represent aminoacids at positions 85-157 of the human PD-L1 amino acid sequence of SEQID NO: 1, i.e., the mature PD-L1 protein from amino acids 19-290 of SEQID NO: 1. Provided are antibodies that bind the STM108 epitope andantibodies that compete for binding to PD-L1 with STM108.

The nucleic acid (DNA) and corresponding amino acid sequences of theheavy and light chain variable (V) domains of the STM073 MAb are shownin Table 3 infra. Table 3 provides both the nucleotide and amino acidsequences of the mature (i.e., not containing the signal peptide) V_(H)and V_(L) domains of STM073 (SEQ ID NOS 2, 3, 10, and 11, respectively)and the V_(H) and V_(L) domain sequences containing the signal peptides(SEQ ID NOS: 87, 88, 89, and 90, respectively). In the heavy chain DNAand protein V domain sequences of the signal sequence containing heavyand light chain domains shown in Table 3, the amino terminal signalsequence (nucleotides 1-58 and amino acids 1-19 of the V_(H) domain andnucleotides 1-66 and amino acids 1-22 of the V_(L) domain, respectively)is represented in italicized font. Also shown in Table 3 are the STM073MAb heavy and light chain V domain CDRs, using both the Kabat andChothia numbering definitions.

In an embodiment, the dual function anti-glycPD-L1 antibody thatspecifically binds glycosylated PD-L1 comprises a V_(H) domain of SEQ IDNO: 3 and a V_(L) domain of SEQ ID NO: 11. In an embodiment, the dualfunction anti-glycPD-L1 antibody competes for specific binding toglycosylated PD-L1 with an antibody comprising a V_(H) domain of SEQ IDNO: 3 and a V_(L) domain of SEQ ID NO: 11. In an embodiment, the dualfunction anti-glycPD-L1 antibody that specifically binds glycosylatedPD-L1 comprises a V_(H) domain comprising Chothia CDRs 1-3 having aminoacid sequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8,respectively, or Kabat CDRs 1-3 having amino acid sequences of SEQ IDNO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively, or a combinationthereof. In an embodiment, the dual function anti-glycPD-L1 antibodycompetes for specific binding to glycosylated PD-L1 with an antibodycomprising a V_(H) domain comprising Chothia CDRs 1-3 having amino acidsequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively,or Kabat CDRs 1-3 having amino acid sequences of SEQ ID NO: 5, SEQ IDNO: 7, and SEQ ID NO: 9, respectively, or a combination thereof. In anembodiment, the dual function anti-glycPD-L1 antibody that specificallybinds PD-L1 comprises a V_(L) domain comprising CDRs 1-3 having aminoacid sequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16,respectively. In an embodiment, the dual function anti-glycPD-L1antibody competes for specific binding to glycosylated PD-L1 with anantibody comprising a V_(L) domain comprising CDRs 1-3 having amino acidsequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16,respectively. In an embodiment, the dual function anti-glycPD-L1antibody that specifically binds glycosylated PD-L1 comprises (a) aV_(H) domain comprising Chothia CDRs 1-3 having amino acid sequences ofSEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively, or KabatCDRs 1-3 having amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 7, andSEQ ID NO: 9, respectively, or a combination thereof; and (b) a V_(L)domain comprising CDRs 1-3 having amino acid sequences of SEQ ID NO: 12,SEQ ID NO: 14, and SEQ ID NO: 16, respectively. In embodiments, theanti-glycPD-L1 antibody competes for specific binding to glycosylatedPD-L1 with an antibody comprising the above-described V_(H) and V_(L)domains and the CDRs therein.

In an embodiment, the dual function anti-glycPD-L1 antibody thatspecifically binds glycosylated PD-L1 comprises a V_(H) domain that is80%, 85%, 90%, 95% 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 3 and/or a V_(L) domain that is 80%, 85%, 90%, 95% 98% or 99%identical to the amino acid sequence of SEQ ID NO: 11, and whichinhibits or blocks binding of glycosylated PD-L1 to PD-1. In anembodiment, the dual function anti-glycPD-L1 antibody that specificallybinds glycosylated PD-L1 comprises a V_(H) domain comprising CDRs 1-3with at least 1, 2, or all 3 CDRs having at least 1, 2, 3, 4 or 5 aminoacid substitutions with respect to the amino acid sequences of SEQ IDNO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively, or amino acidsequences of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively,which dual function anti-glycPD-L1 antibody blocks binding ofglycosylated PD-L1 to PD-1. In an embodiment, the dual functionanti-glycPD-L1 antibody that specifically binds PD-L1 comprises a V_(L)domain comprising CDRs 1-3 with at least 1, 2, or all 3 CDRs having atleast 1, 2, 3, 4 or 5 amino acid substitutions with respect to aminoacid sequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16,respectively. In an embodiment, the anti-glycPD-L1 antibody thatspecifically binds glycosylated PD-L1 comprises (a) a V_(H) domaincomprising CDRs 1-3 with at least 1, 2, or all 3 CDRs having at least 1,2, 3, 4 or 5 amino acid substitutions with respect to the amino acidsequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively,or amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9,respectively; and (b) a V_(L) domain comprising CDRs 1-3 with at least1, 2, or all 3 CDRs having at least 1, 2, 3, 4 or 5 amino acidsubstitutions with respect to amino acid sequences of SEQ ID NO: 12, SEQID NO: 14, and SEQ ID NO: 16, respectively, which antibody blocksbinding of glycosylated PD-L1 to PD-1. Preferably these antibodies havehuman framework regions, i.e., are humanized forms of STM073, andoptionally have human constant domains. Also provided are humanizedforms of STM073 using the AbM, Contact or IMGT defined CDRs, with humanframework regions and, optionally, human constant domains. It will beappreciated by those skilled in the art that one or more amino acidsubstitutions may be made in framework regions and/or the CDRs of ahumanized antibody to improve binding affinity.

In embodiments, the dual function anti-glycPD-L1 antibody competes forspecific binding to glycosylated PD-L1 with an antibody comprising theabove-described V_(H) and V_(L) domains and the CDRs therein. Inembodiments, the anti-glycPD-L1 antibody binds to glycosylated PD-L1with a K_(d) less than half of the K_(d) exhibited relative tounglycosylated PD-L1. In an embodiment, the anti-glycPD-L1 antibodybinds to glycosylated PD-L1 protein with a K_(d) at least 5 times lessthan the K_(d) exhibited relative to unglycosylated PD-L1. In anembodiment, the anti-glycPD-L1 antibody binds to glycosylated PD-L1protein with a K_(d) at least 10 times less than the K_(d) exhibitedrelative to unglycosylated PD-L1 protein. In an embodiment, in a cellflow cytometry binding assay as described in Example 6, the antibodyexhibits binding as expressed as MFI to cells expressing WT PD-L1 thatis 1.5 times, 2 times, 3, times, 4 times, 5 times, 6 times, 7 times, 8times, 9 times or 10 times greater than the MFI for binding to cellsexpressing unglycosylated PD-L1. In an embodiment, the binding affinityof the STM073 antibody, or humanized or chimeric form thereof, orantibody that competes for binding with the foregoing for glycosylatedPD-L1 is from 5-20 nM or from 5-10 nM inclusive of the lower and uppervalues. In an embodiment, the antibody inhibits the interaction of PD-1with PD-L1, and particularly inhibits the interaction of PD-1 expressedby effector T-cells with PD-L1, particularly, glycosylated PD-L1,expressed by tumor cells and also reduces the levels of PD-L1,particularly glycosylated PD-L1, on the surface of the tumor cells,particularly, by increasing the internalization and intracellulardegradation of the PD-L1. Internalization of the PD-L1 expressed ontumor cells following binding by the anti-glycosylated PD-L1 antibodiesprovided herein is a beneficial feature of the anti-glycPD-L1antibodies, as less glycosylated PD-L1 is available on the tumor cellfor interaction with PD-1 on T cells, thereby increasing T cellcytotoxic effector function against the tumor cells and reducing T cellanergy resulting from the PD-L1/PD-1 interaction.

In another particular embodiment, an antibody, or a binding fragmentthereof, is provided that specifically binds glycosylated PD-L1 which isthe anti-glycPD-L1 monoclonal antibody STM108. The nucleic acid (DNA)and corresponding amino acid sequences of the mature heavy and lightchain variable (V) domains (SEQ ID NOs: 18, 19, 26 and 27) of the STM108MAb are shown in Table 3 infra. The DNA and amino acid sequences of theunprocessed heavy chain V domain sequence (i.e., those containing asignal sequence at the N-terminal) are also shown in Table 3 (SEQ IDNOs: 91 and 92) and the amino terminal signal sequence is represented initalicized font (nucleotides 1-57 and amino acids 1-19 of the V_(H)domain). Also shown in Table 3 are the STM108 MAb heavy and light chainV domain CDRs, according to both the Kabat and Chothia definitions.

In an embodiment, the dual function anti-glycPD-L1 antibody thatspecifically and preferentially binds glycosylated PD-L1 comprises aV_(H) domain of the amino acid sequence of SEQ ID NO: 19 and a V_(L)domain of the amino acid sequence of SEQ ID NO: 27. In an embodiment,the anti-glycPD-L1 antibody competes for specific binding toglycosylated PD-L1 with an antibody comprising a V_(H) domain of theamino acid sequence of SEQ ID NO: 19 and a V_(L) domain of the aminoacid sequence of SEQ ID NO: 27. In an embodiment, the anti-glycPD-L1antibody that specifically and preferentially binds glycosylated PD-L1comprises a V_(H) domain comprising Chothia CDRs1-3 having amino acidsequences of SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24,respectively, or Kabat CDRs 1-3 having amino acid sequences of SEQ IDNO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively, or a combinationthereof. In an embodiment, the anti-glycPD-L1 antibody competes forspecific binding to glycosylated PD-L1 with an antibody comprising aV_(H) domain comprising Chothia CDRs 1-3 with amino acid sequences ofSEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24, respectively, or KabatCDRs 1-3 with amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 23, andSEQ ID NO: 25, respectively, or a combination thereof. In an embodiment,the anti-glycPD-L1 antibody that specifically and preferentially bindsglycosylated PD-L1 comprises a V_(L) domain comprising CDRs 1-3 havingamino acid sequences of SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32,respectively. In an embodiment, the anti-glycPD-L1 antibody competes forspecific binding to glycosylated PD-L1 with an antibody comprising aV_(L) domain comprising CDRs 1-3 having amino acid sequences of SEQ IDNO: 28, SEQ ID NO: 30, and SEQ ID NO: 32, respectively. In anembodiment, the anti-glycPD-L1 antibody that specifically andpreferentially binds glycosylated PD-L1 comprises (a) a V_(H) domaincomprising Chothia CDRs1-3 having amino acid sequences of SEQ ID NO: 20,SEQ ID NO: 22, and SEQ ID NO: 24, respectively, or Kabat CDRs 1-3 havingamino acid sequences of SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25,respectively, or a combination thereof; and (b) a V_(L) domaincomprising CDRs 1-3 having amino acid sequences of SEQ ID NO: 28, SEQ IDNO: 30, and SEQ ID NO: 32, respectively. In embodiments, theanti-glycPD-L1 antibody competes for specific binding to glycosylatedPD-L1 with an antibody comprising the above-described VH and VL domainsand the CDRs therein.

In an embodiment, the anti-glycPD-L1 antibody that specifically andpreferentially binds glycosylated PD-L1 comprises a V_(H) domain that is80%, 85%, 90%, 95% 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 19 and a V_(L) domain that is 80%, 85%, 90%, 95% 98% or 99%identical to the amino acid sequence of SEQ ID NO: 27. In an embodiment,the anti-glycPD-L1 antibody that specifically and preferentially bindsglycosylated PD-L1 comprises a V_(H) domain comprising CDRs 1-3 with atleast 1, 2, or all 3 CDRs having at least 1, 2, 3, 4 or 5 amino acidsubstitutions with respect to the amino acid sequences of SEQ ID NO: 20,SEQ ID NO: 22, and SEQ ID NO: 24, respectively, or CDRs 1-3 having aminoacid sequences of SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25,respectively, or a combination thereof. In an embodiment, theanti-glycPD-L1 antibody that specifically and preferentially bindsglycosylated PD-L1 comprises a V_(L) domain comprising CDRs 1-3 with atleast 1, 2, or all 3 CDRs having at least 1, 2, 3, 4 or 5 amino acidsubstitutions with respect to the amino acid sequences of SEQ ID NO: 28,SEQ ID NO: 30, and SEQ ID NO: 32, respectively. In an embodiment, theanti-glycPD-L1 antibody that specifically and preferentially bindsglycosylated PD-L1 comprises (a) a V_(H) domain comprising CDRs 1-3 withat least 1, 2, or all 3 CDRs having at least 1, 2, 3, 4 or 5 amino acidsubstitutions with respect to the amino acid sequences of SEQ ID NO: 20,SEQ ID NO: 22, and SEQ ID NO: 24, respectively, or with respect to theamino acid sequences of SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25,respectively, or a combination thereof; and/or (b) a V_(L) domaincomprising CDRs 1-3 with at least 1, 2, or all 3 CDRs having at least 1,2, 3, 4 or 5 amino acid substitutions with respect to the amino acidsequences of SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32,respectively.

Also provided are humanized forms of STM108 using the AbM, Contact orIMGT defined CDRs, with human framework regions and, optionally, humanconstant domains. Preferably these antibodies have human frameworkregions, i.e., are humanized forms of STM108, and optionally have humanconstant domains.

In embodiments, the anti-glycPD-L1 antibody competes for specificbinding to glycosylated PD-L1 with an antibody comprising theabove-described V_(H) and V_(L) domains and the CDRs therein. Thehumanized forms of STM108 may have one or more amino acid substitutions,deletions or insertions in the CDRs and/or framework regions thatimprove one or more properties of the antibody, such as antigenaffinity. In embodiments, the anti-glycPD-L1 antibody binds toglycosylated PD-L1 with a K_(d) less than half of the K_(d) exhibitedrelative to unglycosylated PD-L1. In an embodiment, the anti-glycPD-L1antibody binds to glycosylated PD-L1 protein with a K_(d) at least 5times less than the K_(d) exhibited relative to unglycosylated PD-L1. Inan embodiment, the anti-glycPD-L1 antibody binds to glycosylated PD-L1protein with a K_(d) at least 10 times less than the K_(d) exhibitedrelative to unglycosylated PD-L1 protein. In an embodiment, in a cellflow cytometry binding assay as described in Example 6, the antibodyexhibits binding as expressed as MFI to cells expressing WT PD-L1 thatis 1.5 times, 2 times, 3, times, 4 times, 5 times, 6 times, 7 times, 8times, 9 times or 10 times greater than the MFI for binding to cellsexpressing unglycosylated PD-L1. In an embodiment, the binding affinityof STM108 MAb, or chimeric or humanized form thereof, for glycosylatedPD-L1 is from 5-20 nM or from 5-10 nM inclusive of the lower and uppervalues. In an embodiment, the antibody inhibits the interaction of PD-1with PD-L1, and particularly inhibits the interaction of PD-1 expressedby effector T-cells with PD-L1, particularly, glycosylated PD-L1,expressed by tumor cells. The antibody preferably has human frameworkregions and in certain embodiments a human constant domain.

Also encompassed by the present embodiments is an isolated dual functionanti-glycPD-L1 monoclonal antibody, or chimeric or humanized formthereof, that binds an epitope within the human PD-L1 amino acidsequence DAGVYRCMISYGGADYKRITV (SEQ ID NO: 86), which is a peptideportion of the human PD-L1 sequence of SEQ ID NO: 1. In an embodiment,the epitope bound by the antibody includes non-contiguous amino acids,and the epitope is a conformational epitope. In an embodiment, theantibody specifically binds a human PD-L1 epitope corresponding tonon-contiguous amino acid residues encompassing positions Y112, R113 andS117 of the human PD-L1 amino acid sequence SEQ ID NO: 1 shown as theunderlined amino acid residues in the following sequence:DAGVYRCMISYGGADYKRITV (SEQ ID NO: 86).

Provided in another aspect is an isolated dual function anti-glycPD-L1antibody, e.g., a monoclonal antibody, or a binding fragment thereof,particularly a humanized or human antibody, that specifically binds toan epitope within an amino acid sequence selected fromVHGEEDLKVQH------DAGVYRCMISYGGADYKRITV (SEQ ID NO: 85), orDAGVYRCMISYGGADYKRITV (SEQ ID NO: 86), which sequence is located withinthe mature human PD-L1 polypeptide sequence of SEQ ID NO: 1. Theantibody preferably has human framework regions and in certainembodiments a human constant domain. Provided in another aspect is anisolated dual function anti-glycPD-L1 antibody, e.g., a monoclonalantibody, or a binding fragment thereof, particularly a humanized orhuman antibody, that specifically binds to an epitope within the aminoacid sequence LKVQHSSYRQR------EGYPKAEVIWTSSDHQ, which are amino acids74 to 84 and 158 to 173 of SEQ ID NO:1. The antibody preferably hashuman framework regions and in certain embodiments a human constantdomain

In a certain embodiment, an isolated anti-glycPD-L1 antibody,particularly a monoclonal, humanized, or human antibody, that binds anepitope that comprises amino acid residues H69, Y112, R113 and K124 ofSEQ ID NO: 1 is provided. In an aspect, an isolated anti-glycPD-L1antibody is provided that specifically binds glycosylated human PD-L1,such that when bound to human PD-L1, the antibody binds at least one ofthe following amino acid residues: H69, Y112, R113 and K124 of SEQ IDNO: 1. In another embodiment, an isolated anti-glycPD-L1 antibody,particularly a monoclonal, humanized, or human antibody, that binds anepitope that comprises amino acid residues S80, Y81, K162 and S169 ofSEQ ID NO: 1 is provided. In an aspect, an isolated anti-glycPD-L1antibody is provided that specifically binds glycosylated human PD-L1,such that when bound to human PD-L1, the antibody binds at least one ofthe following amino acid residues: S80, Y81, K162 and S169 of SEQ ID NO:1.

In another aspect, an isolated dual function anti-glycPD-L1 antibody,particularly a monoclonal, humanized, or human antibody, is providedthat specifically binds glycosylated human PD-L1, such that when boundto human PD-L1, the antibody binds at least one of the following aminoacid residues: Y112, R113 and S117 of SEQ ID NO: 1.

Provided in another aspect is an isolated dual function anti-glycPD-L1antibody, particularly a monoclonal, humanized, or human antibody, thatspecifically binds glycosylated human PD-L1, such that when bound tohuman PD-L1, the antibody binds at least one amino acid within the aminoacid region from V68 to V128 of SEQ ID NO: 1 or within the amino acidregion from D108 to V128 of SEQ ID NO: 1. In an aspect, an isolated dualfunction anti-glycPD-L1 antibody is provided that specifically bindsglycosylated human PD-L1, such that when bound to human PD-L1, themonoclonal antibody binds the following group of amino acid residues:H69, Y112, R113 and K124 within the amino acid region from V68 to V128of SEQ ID NO: 1. In an aspect, an isolated dual function anti-glycPD-L1antibody is provided that specifically binds glycosylated human PD-L1,such that when bound to human PD-L1, the monoclonal antibody binds thefollowing group of amino acid residues: Y112, R113, S117 within theamino acid region from D108 to V128 of SEQ ID NO: 1.

In an aspect, the dual function anti-glycPD-L1 antibody binds within theregion of L74 to R84 and/or E158 to Q173 of SEQ ID NO:1, and preferably,one or more of residues S80, Y81, K162 and S169 of SEQ ID NO: 1.

Provided in another aspect is an isolated dual function anti-glycPD-L1antibody, or a binding fragment thereof, particularly a monoclonal,humanized, or human antibody, that specifically binds glycosylated humanPD-L1 protein versus non-glycosylated, such that when bound to humanPD-L1, the antibody binds at least amino acid region V68-V128 or atleast amino acid region D108-V128 of the PD-L1 protein (SEQ ID NO: 1),or a combination thereof. The antibody preferably has human frameworkregions and in certain embodiments a human constant domain.

Provided in another aspect is an isolated dual function anti-glycPD-L1antibody or a binding fragment thereof, comprising (a) a V_(H) domaincomprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, SEQID NO: 21, SEQ ID NO: 4 or SEQ ID NO: 20; a CDR2 comprising an aminoacid sequence of SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 6 or SEQ ID NO:22; and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9, SEQ IDNO: 25, SEQ ID NO: 8 or SEQ ID NO: 24; and (b) a V_(L) domain comprisinga CDR1 comprising an amino acid sequence of SEQ ID NO: 12 or SEQ ID NO:28; a CDR2 comprising an amino acid sequence of SEQ ID NO: 14 or SEQ IDNO: 30; and a CDR3 comprising an amino acid sequence of SEQ ID NO: 16 orSEQ ID NO: 32. In an embodiment, the antibody is a humanized antibodythat has human framework regions, and, optionally, a human antibodyconstant domain.

Provided in another aspect is an isolated nucleic acid moleculecomprising a nucleotide sequence selected from SEQ ID NOs: 2 or 18,which encodes a V_(H) domain and/or a nucleotide sequence selected fromSEQ ID NO: 10 or 26, which encodes a V_(H) domain. An embodimentprovides an isolated nucleotide sequence encoding a V_(H) domain of adual function anti-glycPD-L1 antibody, wherein the nucleotide sequenceis at least 90-98% identical to the nucleotide sequence of SEQ ID NOs: 2or 18. Another embodiment provides an isolated nucleotide sequenceencoding a V_(L) domain of a dual function anti-glycPD-L1 antibody,wherein the nucleotide sequence is at least 90-98% identical to thenucleotide sequence of SEQ ID NOs: 19 or 26. In embodiments, thenucleotide sequences encoding the V_(H) and/or the V_(L) domains are90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical toSEQ ID NOs: 2 or 18, or SEQ ID NOs: 10 or 26, respectively.

In certain aspects, the dual function anti-glycPD-L1 antibody is an IgG,IgM, IgA, an isotype thereof, such as IgG1, IgG2a, IgG2b, IgG4, or anantigen binding fragment thereof. In other aspects, anti-glycPD-L1antibody is an Fab′, a F(ab′)2, a F(ab′)3, a monovalent scFv, a bivalentscFv, a bispecific antibody, a bispecific scFv, or a single domainantibody. In some aspects, the anti-glycPD-L1 antibody is a chimericantibody, human antibody or a humanized antibody. In an aspect, the dualfunction anti-glycPD-L1 antibody is recombinantly produced. In furtheraspects, the dual function anti-glycPD-L1 antibody is conjugated to animaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

In an aspect, a composition comprising a dual function anti-glycPD-L1antibody (e.g., an antibody that selectively and preferentially binds toglycosylated PD-L1 relative to unglycosylated PD-L1 and effectuatesinternalization and/or degradation of PD-L1 as described herein) in apharmaceutically acceptable carrier or medium is provided.

Provided in a further aspect is an isolated polypeptide comprising apeptide of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more) contiguous amino acids of human PD-L1 comprisingat least one amino acid corresponding to position N35, N192, N200 orN219 within the extracellular domain (ECD) of human PD-L1. In anembodiment, the isolated polypeptide comprises a peptide of at least 7(e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore) contiguous amino acids of human PD-L1, comprising at least oneamino acid corresponding to position N35, N192, N200 or N219 of humanPD-L1, in which at least one of the amino acids corresponding toposition N35, N192, N200 or N219 of PD-L1 is glycosylated. In anembodiment, the isolated polypeptide is fused or conjugated to animmunogenic polypeptide (e.g., keyhole limpet hemocyanin, KLH). Incertain aspects, the polypeptide further comprises a cysteine (Cys)residue at its amino (N)- or carboxy (C)-terminus. For example, thepolypeptide may be conjugated to an immunogenic polypeptide by adisulfide linkage at the Cys residue. In a particular embodiment, thePD-L1 peptide comprising at least one amino acid residue glycosylated ata position corresponding to position N35, N192, N200 or N219 of humanPD-L1 is used as an immunogen to generate anti-glycPD-L1 antibodies.

In a further aspect, a composition is provided comprising a polypeptidecomprising a peptide of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more) contiguous amino acids of humanPD-L1 comprising at least one amino acid corresponding to position N35,N192, N200 or N219 within the ECD of human PD-L1, wherein at least oneof said amino acids corresponding to position N35, N192, N200 or N219 ofPD-L1 is glycosylated, wherein the polypeptide is formulated in apharmaceutically acceptable carrier, diluent, excipient, or vehicle.

In yet a further aspect, an immunogenic composition is providedcomprising a polypeptide comprising a peptide fragment of at least 7contiguous amino acids of human PD-L1, which comprises at least oneamino acid corresponding to position N35, N192, N200 or N219 of thehuman PD-L1 polypeptide, wherein at least one of the amino acidscorresponding to position N35, N192, N200 or N219 within the ECD of thePD-L1 polypeptide is glycosylated, and wherein the polypeptide isformulated in a pharmaceutically acceptable carrier, diluent, excipient,or vehicle. In some aspects, the immunogenic composition furthercomprises an adjuvant, such as alum or Freund's adjuvant.

Provided in a further aspect is a method for treating a subject in need,such as a subject with a cancer, which comprises administering to thesubject an effective amount of a dual function anti-glycPD-L1 antibodyas described herein. In specific embodiments, the anti-glycPD-L1antibody is a humanized or chimeric form of one of MAbs STM073 orSTM108. In other embodiments, the dual function anti-glycPD-L1 antibodyis an antibody that competes for binding to glycosylated PD-L1 with MAbSTM073 or MAb STM108. In an embodiment, a method of treating a cancer ina subject in need thereof, is provided in which the method comprisesadministering an effective amount of an antibody as described herein tothe subject. In certain preferred embodiments, the cancer cells arepositive for PD-L1, particularly glycosylated PD-L1. The cancer cellsmay also be positive for one or more other cancer cell markers, such as,but not limited to, EGFR. In nonlimiting embodiments, the cancer,disease or pathology to be treated in the subject is a breast cancer,lung cancer, head & neck cancer, prostate cancer, esophageal cancer,tracheal cancer, skin cancer brain cancer, liver cancer, bladder cancer,stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer,cervical cancer, testicular cancer, colon cancer, rectal cancer or skincancer. In certain embodiments, the cancer to be treated is an adrenalcancer, an anal cancer, a bile duct cancer, a bone cancer, a brain/CNStumor in an adult, a brain/CNS tumor in a child, a breast cancer in aman, cancer in an adolescent, cancer in a child, cancer in a youngadult, cancer of unknown primary, Castleman disease, cervical cancer,endometrial cancer, Ewing family tumor, eye cancer, gallbladder cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),gestational trophoblastic disease, Hodgkin's disease, Kaposi sarcoma,kidney cancer, laryngeal or hypopharyngeal cancer, leukemia (e.g., adultacute lymphocytic (ALL), acute myeloid (AML), chronic lymphocytic (CLL),chronic myeloid (CML), chronic myelomonocytic (CMML), childhoodleukemia), lung cancer (e.g., non-small cell, small cell), lungcarcinoid tumor, lymphoma, lymphoma of the skin, malignant mesothelioma,multiple myeloma, myelodysplastic syndrome, naval cavity cancer,paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,non-Hodgkin lymphoma, non-Hodgkin lymphoma in a child, oral cavitycancer, oropharyngeal cancer, osteosarcoma, penile cancer, pituitarytumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivarygland cancer, sarcoma (e.g., adult soft tissue cancer), skin cancer(e.g., basal and squamous cell, melanoma, merkel cell), small intestinecancer, stomach cancer, testicular cancer, thymus cancer, thyroidcancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrommacroglobulinemia, or Wilms tumor.

In certain aspects, the anti-glycPD-L1 antibody is formulated in apharmaceutically acceptable composition. In further aspects, theantibody is administered systemically or parenterally. In particularaspects, the antibody is administered intravenously, intradermally,intratumorally, intramuscularly, intraperitoneally, subcutaneously,intrathecally, or locally. In other aspects, one or more than one typeof dual function anti-glycPD-L1 antibody may be co-administered to asubject in need. Co-administration of antibodies may involve theadministration of one antibody before, after, or concurrently withanother antibody.

In an aspect, a method of treating a subject who has a cancer or tumor,particularly a cancer or tumor that expresses PD-L1 on the cancer ortumor cell surface is provided. Such a method comprises administering tothe subject in need thereof an effective amount of a dual functionanti-glycPD-L1 antibody to inhibit or block the interaction of PD-L1with PD-1 and prevent immunosuppression and promote killing of thecancer or tumor cells by the subject's effector T lymphocytes. Inembodiments, the anti-glycPD-L1 antibody is a chimeric or humanized formof STM073 or STM108, or an antibody that competes with one or both ofthese antibodies for selectively binding to glycosylated PD-L1.

In some aspects, the methods comprise administering at least a secondanticancer therapy or drug to the subject who is receiving treatmentwith an anti-glycPD-L1 antibody. The second anticancer therapy mayconstitute, without limitation, a surgical therapy, chemotherapy,radiation therapy, cryotherapy, hormonal therapy, immunotherapy orcytokine therapy. The second anticancer drug is also not intended to belimited and will be able to be practically or empirically determined bythe clinician, medical professional (e.g., oncologist) skilled in theart. As will be appreciated by one having skill in the art, theadministration of at least a second anticancer therapy or drug may occurbefore, after, or simultaneously with the administration of a dualfunction anti-glycPD-L1 antibody.

Provided in another aspect is a method of determining if a patient withcancer is likely to benefit from treatment with (i.e., is a candidatefor treatment with) an agent that blocks binding of PD-L1 with PD-1, themethod comprising testing for the presence of glycosylated PD-L1 oncells derived from a sample of the patient's cancer cells using anantibody as described herein, wherein detecting the binding of theantibody to the patient's cancer cells indicates that the patient is acandidate for therapy. The method may further comprise administering tothe patient an effective amount of an agent that prevents binding ofPD-L1 to PD-1 if subject's cancer cells are found to be positive for theexpression of glycosylated PD-L1 protein. In an embodiment, the agentthat prevents binding of PD-L1 to PD-1 is an anti-glycPD-L1 antibody,preferably a dual function anti-glycPD-L1 antibody, an anti-PD-1antibody, or a combination thereof. In an embodiment, the anti-glycPD-L1antibody and/or anti-PD-1 antibody is administered in combination withanother anti-cancer drug or therapeutic. In an embodiment, ananti-glycPD-L1 antibody is administered in combination with anotheranti-glycPD-L1 antibody, such as described herein. In an embodiment, themethod further involves obtaining the cancer cell sample from thepatient.

Provided in yet another aspect is a method for assessing glycosylation,N-linked glycosylation, or N-glycosylation of PD-L1 protein in abiological sample, in which the method comprises contacting thePD-L1-containing sample with an antibody as described herein. In someaspects, the method is an in vitro method or assay. In certain aspects,the biological sample is cell sample, a tissue sample, a body fluid(e.g., plasma, serum, blood, urine, sputum, lymph, ascites fluid,intraperitoneal fluid, cerebral or spinal fluid, and the like). Inparticular embodiments, the sample is a cell sample or a cell samplefrom a tumor or cancer obtained from a subject having a cancer or tumor.Such a cancer or tumor cell sample may be assayed for glycosylated PD-L1on the cancer or tumor cell surface using the dual functionanti-glycPD-L1 antibodies as described herein, particularly to determinethat, if glycosylated PD-L1 is present on the subject's cancer or tumorcells, then the cells would likely be appropriate targets for treatmentwith the anti-glycPD-L1 antibodies as described.

Provided in another aspect is a method of making an antibody in whichthe method comprises administering to a subject (e.g., an animal) apolypeptide according to the embodiments (e.g., a polypeptide comprisinga fragment of at least 7 contiguous amino acids of human PD-L1comprising at least one amino acid corresponding to position N35, N192,N200 or N219 of human PD-L1, wherein at least one of said amino acidscorresponding to position N35, N192, N200 or N219 of PD-L1 isglycosylated) and isolating the antibody from the subject. By way ofexample, the animal can be a non-human primate, mouse, rat, rabbit,goat, or a human. In certain aspects a method further comprisesidentifying the CDRs of the antibody and humanizing the sequences (i.e.,framework sequences) surrounding the CDRs to produce a humanizedantibody using methods and procedures known in the art. In still furtheraspects, the method comprises recombinantly expressing the humanizedantibody. Thus, in a further embodiment, there is provided an isolatedantibody produced by the foregoing method. Thus, in some embodiments,there is provided an isolated antibody that selectively binds to anepitope, such as a conformational epitope, of a glycosylated PD-L1polypeptide (e.g., a polypeptide comprising a fragment of at least 7contiguous amino acids of human PD-L1 comprising at least one amino acidcorresponding to position N35, N192, N200 or N219 of human PD-L1,wherein at least one of said amino acids corresponding to position N35,N192, N200 or N219 of PD-L1 is glycosylated) relative to unglycosylatedPD-L1. Another aspect provides a method for immunizing a subject toproduce an immune response, e.g., an antibody response directed againstan antigen, comprising administering an effective amount of apolypeptide of the embodiments (e.g., a glycosylated PD-L1 polypeptide)as immunogenic antigen to the subject.

In specific embodiments, the invention provides methods of makingbiparatopic antibodies that have two different antigen binding domainsthat each bind an epitope that does not overlap with the epitope of theother antigen binding domain of glycosylated PD-L1, and at least onebinding domain (and in certain embodiments, both binding domains) bindspreferentially to a glycosylated form of PD-L1, for example by bindingan epitope containing one or more of the glycosylation sites listedherein. These antibodies can crosslink cell surface proteins promotinginternalization, lysosomal trafficking and degradation. Such biparatopicantibodies may be generated by screening for antibodies thatpreferentially bind to a glycosylated PD-L1 protein relative to anon-glycosylated form of PD-L1 using screening methods known in the artand also as described in co-pending provisional application No.62/361,298, identifying two antibodies that bind non-overlappingepitopes of glycosylated PD-L1, where preferably one or bothpreferentially bind the glycosylated form of PD-L1 as compared to thenon-glycosylated form. Antibodies against any of the glycosylationcontaining epitopes or epitopes described herein to which antibodiespreferentially bind to glycosylated PD-L1 can be used. The two antigenbinding domains can be arranged in an antibody molecule, for example, asdescribed in Dimasi et al., J. Mol. Biol., 393:672-692 (2009). Inspecific embodiments, one of the antigen binding domains is engineeredto be in the format of a single chain Fv which is then linked to the Nterminus of the heavy and/or light chains of an antibody having theother antigen binding domain or to the C-terminus of the CH3 domain,e.g., via a peptide linker.

In another aspect, antibody-drug conjugates (ADCs) are provided, inwhich the anti-glycPD-L1 antibodies as described herein, in particular,those that show effective internalization activity following binding toglycosylated PD-L1, are chemically linked to antineoplastic drugs andagents to produce an anti-glycPD-L1 antibody-drug conjugate, asdescribed and exemplified herein. In embodiments, the anti-glycPD-L1 MAbADCs are highly effective in killing tumor or cancer cells and inantineoplastic therapies for treating subjects with cancer. Inembodiments, the anti-glycPD-L1 antibody component of the ADC is abispecific, multispecific, biparatopic, or multiparatopic antibody, oran antigen binding portion thereof. In other embodiments, theanti-glycPD-L1 antibody is chemically linked to an antimitotic agent,such as a maytansine derivative, e.g., a maytansinoid such as DM1 orDM4, or to a tubulin-polymerizing auristatin, e.g., monomethylauristatin E (MMAE) or monomethyl auristatin F (MMAF), as describedfurther herein. In an embodiment, the linker to the anti-glycPD-L1antibody is stable in extracellular fluid, but is cleaved by cathepsinonce the ADC has entered a tumor or cancer cell, thus activating theantimitotic mechanism of MMAE or other toxin drug. In an embodiment, theantibody component of the ADC is STM073 MAb or STM108 MAb as describedherein. In an embodiment, the STM108 MAb-containing ADC (STM108-ADC) ischemically linked to MMAE via a cleavable linker. In a particularembodiment, the STM108-ADC comprises a structure in which the STM108 MAbis chemically linked via cysteine residues in its C-region to amaleimide and caproic acid (MC) attachment group, which is chemicallylinked to a cathepsin-cleavable linker, such as “vc” consisting ofvaline (Val) and citruline (Cit), which is chemically attached to thespacer “PAB”, i.e., paraminobenzoic acid, which is chemically linked toMMAE cytotoxin, thus producing the ADC, designated by its componentstructure STM108-MC-vc-PAB-MMAE.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the embodimentsdescribed herein without being limiting. The patent or application filecontains at least one drawing executed in color. Copies of this patentor patent application publication with color drawing(s) will be providedby the Office upon request and payment of the necessary fee.

FIGS. 1A-1H. PD-L1 is Glycosylated in Cancer Cells. 1A. Expression ofPD-L1 protein in primary breast cancer patient samples. Western blotanalysis of PD-L1 in representative breast cancer patient samples. 1B.Western blot analysis of PD-L1 in four representative breast cancer celllines, four melanoma cell lines, three lung cancer cell lines and threecolon cancer cell lines. 1C. Western blot analysis of PD-L1 expressionin shCTRL and two independent shPD-L1 stable clones of A431 cells. PD-L1was transiently transfected into the shPD-L1#5 clone. D. Glycoproteinstaining of purified PD-L1 protein with or without PNGase F treatment.The Coomassie blue stained panel represents the total amount of PD-L1protein. The upper bands which appear in lanes 4 and 5 are from theloading of PNGase F. (−) Ctrl, a control for non-glycoprotein; (+) Ctrl,a control for glycoprotein. E. Glycosylation pattern of PD-L1-GFP,HA-PD-L1, and PD-L1-Flag proteins. Cell lysates were treated with PNGaseF and Endo H and analyzed by Western blot. F. GFP-tagged PD-L1 fulllength (WT), extracellular domain (ECD), or intracellular domain (ICD)was transiently expressed in 293T cells. Cells were then treated with orwithout 5 μg/ml tunicamycin (TM) overnight. Protein expression of PD-L1was examined using Western blot. G. Schematic diagram of arepresentative PD-L1 protein expression construct as used in theexperimental studies described herein, showing the full-length PD-L1protein and its component extracellular domain (ECD), intracellulardomain (ICD), signal peptide (SP), transmembrane domain (TM). In thediagram, four N-glycosylation sites (NXT motifs) in the ECD domain ofPD-L1 are shown in red. The numbers indicate the positions of the aminoacid residues in the PD-L1 polypeptide. H. Western blot analysis of theprotein expression pattern of PD-L1 WT and glycosylation mutants (NQmutants) of PD-L1. Lane 14 indicates non-glycosylated, wild-type (WT)PD-L1 treated overnight with tunicamycin (TM). In the figures, the blackcircles indicate glycosylated PD-L1, and the black arrowheads indicatenon-glycosylated PD-L1.

FIGS. 2A-2G. Glycosylation Stabilizes PD-L1 Expression and is Requiredfor Cancer Cell-associated Immunosuppression. A and B: Results ofWestern blot analyses of PD-L1 protein in PD-L1-Flag expressing 293Tcells. Cells were treated with 20 mM cycloheximide (CHX) (A) and 5 μMMG132, a proteosome inhibitor (B), at the indicated intervals andanalyzed by Western blot. The intensity of the PD-L1 protein wasquantified using a densitometer. C. Western blot analysis of proteinstability of PD-L1WT, and PD-L1 glycosylation mutants N35Q, N192Q,N200Q, N219Q, and 4NQ determined as described in (A). Beneath theWestern blot analysis result: Quantification of protein half-life ofPD-L1 WT and the four NQ mutants via densitometry. D. Interaction ofPD-1 and PD-L1 proteins with or without TM or anti-PD-L1 antibodytreatment. Confocal image shows bound PD-1/Fc fusion protein on membraneof PD-L1 WT expressing 293T cells (staining panels on left).Quantification of bound PD-1 protein in PD-L1/PD-1 interaction assay(plot on right). E. Binding affinity of PD-1 with PD-L1 WT or 4NQproteins. The lysates of PD-L1 WT or 4NQ expressing 293T cells wereincubated with or without PD-1/Fc fusion protein. The PD-L1 proteinswere then immunoprecipitated with anti-Flag antibody and analyzed byWestern blot. F. Co-immunoprecipitation measuring the interaction ofPD-1 and PD-L1 in PD-L1 WT or 4NQ expressing 293T cells. G. Levels ofsoluble IL-2 expression in co-cultures of Jurkat T-cells and PD-L1 WT or4NQ expressing cells. Cells were pretreated with MG132 prior to theexperiments shown in (D), (E) and (G). In the figure, black circlesindicate glycosylated PD-L1; arrowheads indicate non-glycosylated PD-L1;TM: tunicamycin; * indicates statistically significant by Student's ttest. All error bars are expressed as mean±SD of 3 independentexperiments.

FIGS. 3A-3D. Expression of PD-L1 Protein in Cancer Cells. A. Westernblot analysis of PD-L1 in lung cancer cells. B. Western blot analysis ofPD-L1 in colon cancer cells. C. Western blot analysis of PD-L1 in breastcancer cells. D. Western blot analysis of PD-L1 in ovarian cancer cells.Black circles=glycosylated PD-L1; arrowheads=, non-glycosylated PD-L1.

FIGS. 4A-4D. PD-L1 is Glycosylated in Cancer Cells. A. Western blotanalysis of PD-L1 in cancer cells using different anti-PD-L1 antibodies.Four BLBC cell lines, HCC1937, SUM149, MB-231 and BT20, and two non-BLBCcell lines, MB-483 and MB-474 were selected to analyze the expression ofPD-L1 using different antibodies. B. Western blot analysis of PD-L1 inshCTRL and two independent shPD-L1 stable clones of MDA-MB-231 and A431cells. C. Schematic diagram of dual-expression construct for Flag-PD-L1and shRNA of PD-L1. D. Glycosylation pattern of PD-L1 protein inMDA-MB-231 and A431 cells. Cell lysates were treated with PNGase F andanalyzed by Western blot. Black circle=glycosylated PD-L1;arrowhead=non-glycosylated PD-L1.

FIGS. 5A-5E. Expression of Glycosylated and Non-glycosylated PD-L1Protein. A. Western blot analysis of PD-L1-Myc, PD-L1-Flag, and HA-PD-L1proteins in tunicamycin (TM) treated cells. B. Western blot analysis ofPD-L1-GFP-WT, PD-L1-GFP-ECD and PD-L1-GFP-ICD proteins in tunicamycin(TM) treated or untreated cells. C. Western blot analysis of PD-L1-Myc,PD-L1-Flag, HA-PD-L1, PD-L1-GFP-WT, PD-L1-GFP-ECD and PD-L1-GFP-ICDproteins in tunicamycin (TM) treated cells. The intensity ofglycosylated PD-L1 protein (black bars) or non-glycosylated PD-L1protein (red bars) was determined by a densitometry quantification (inbar graph below Western blot analysis). D. The mean of the intensity ofglycosylated PD-L1 protein (black bar) or non-glycosylated PD-L1 protein(red bar) obtained from the bar graph shown in (C) above. Error barsrepresent standard deviation (SD). E. Glycosylation pattern of PD-L1protein in PD-L1 expressing HEK 293T cells. Cell lysates were treatedwith or without PNGase F or O-glycosidase and analyzed by Western blot.Black circle=glycosylated PD-L1; arrowhead=non-glycosylated PD-L1.Tunicamycin is a nucleoside antibiotic that inhibits N-linkedglycosylation of proteins.

FIGS. 6A and 6B. N-glycosylation Sites of PD-L1 Protein. A sequencealignment of the PD-L1 amino acid sequences from different species. FourNXT motifs, N35, N192, N200, and N219 are boxed and presented in red,and two non-NXT motifs, N63 and N204 are presented in green. Redbox=conserved NXT motif. FIG. 6A: Consensus sequence (SEQ ID NO: 74);Q9NZQ7_HUMAN (SEQ ID NO: 75); Q9EP73_MOUSE (SEQ ID NO: 76); D4AE25_RAT(SEQ ID NO: 77); C5NU11_BOVINE (SEQ ID NO: 78); Q4QTK1_PIG (SEQ ID NO:79); and F7DZ76_HORSE (SEQ ID NO: 80). FIG. 6B: Consensus sequence (SEQID NO: 94); Q9NZQ7_HUMAN (SEQ ID NO: 95); Q9EP73_MOUSE (SEQ ID NO: 96);D4AE25_RAT (SEQ ID NO: 97); C5NU11_BOVINE (SEQ ID NO: 98); Q4QTK1_PIG(SEQ ID NO: 99); and F7DZ76_HORSE (SEQ ID NO: 100).

FIGS. 7A-7H. LC-MS/MS-based Identification of N-glycopeptides.LC-MS/MS-based identification of N-glycopeptides corresponding to eachof the four N-glycosylation sites, N35 (A and E), N192 (B and F), N200(C and G), and N219 (D and H) of PD-L1 from HEK 293 cells. The LC-MSprofiles (A-D) are shown as spectra averaged over a period of elutiontime (as labeled in figures) when a representative subset of glycoformswere detected. For each N-glycosylation site, one representive HCD MS²spectrum (E-H) is shown to exemplify its identification based ondetection of y1 ion (tryptic peptide backbone carrying the GlcNAcattached to the N-glycosylated Asn), along with the b and y ionsdefining its peptide sequence. The cartoon symbols used for the glycans(see inset) conform to the standard representation recommended by theConsortium for Functional Glycomics: Additional Hex and HexNAc weretentatively assigned as either lacNAc (Gal-GlcNAc) or lacdiNAc(GalNAc-GlcNAc) extension from the trimannosyl core (Man₃-GlcNAc₂),which can either be core fucosylated or not. The sequences depicted inFIGS. 7E-7H are set forth in SEQ ID NOs: 81-84, respectively.

FIGS. 8A-8H. PD-L1 Glycosylation Changes Protein Stability andImmunosuppression Function of PD-L1. A-B. Western blot analysis of PD-L1protein in PD-L1-Flag expressing HEK 293T cells. Cells treated with orwithout tunicamycin were treated with 20 mM cycloheximide (CHX) (A) and5 μM MG132 (B), for the indicated intervals (hr) and analyzed by Westernblot. C. Western blot analysis of PD-L1 in A431 cells either treated ornot treated with tunicamycin and treated with 20 mM cycloheximide (CHX)for the designated times. The bottom panel shows a densitometryquantification of glycosylated versus non-glycosylated PD-L1 protein. D.Schematic diagram of a PD-L1/PD-1 interaction assay. E. Confocal imagesshowing membrane localized PD-L1 WT or 4NQ proteins in A431 cells. F.Membrane localization of PD-L1 WT or PD-L1 4NQ proteins. Afterbiotinylation of membrane localized PD-L1 WT or 4NQ proteins, thebiotinylated proteins were pull-downed by streptavidin agarose. Membranelocalized PD-L1 WT or 4NQ proteins were examined by Western blot. Theratio of membrane bound PD-L1 WT or 4NQ protein obtained fromdensitometry quantification of samples is shown beneath the blot. G. Theinteraction of PD-1 and PD-L1 proteins in PD-L1 WT-, N35Q-, N192Q-,N200Q-, N219Q-, or 4NQ-expressing cells. All error bars are expressed asmean±SD of 3 independent experiments. H. Schematic diagram of an ELISAdesigned to detect IL-2 production following T-cell/tumor cellinteraction via PD-1/PD-L1, respectively. PD-L1-expressing tumor cloneswere co-cultured with Jurkat T-cells that overexpressed PD-1. IL-2production from Jurkat cells was measured using ELISA, as set forth,e.g., in Examples 1, 4, and 5 infra. In A-C, black circle=glycosylatedPD-L1; arrow head=non-glycosylated PD-L1.

FIGS. 9A-9F. Glycosylation of PD-L1 is Required for CancerCell-associated Immunosuppression. A. Flow cytometry measuring theinteraction of membrane bound PD-1 and PD-L1 WT or PD-L1 4NQ mutantprotein expressed on BT549 cells. Cells were pretreated with MG132 priorto experiment. B. Time lapse microscopy image showing the dynamicinteraction between PD-L1 and PD-1 at the last time point. Redfluorescent (nuclear restricted RFP) and green fluorescent (greenfluorescent labeled PD-1/Fc protein) merged images (20×) of PD-L1 WT or4NQ expressing cells. The kinetic graph at right in (B) shows thequantitative binding of the PD-1/Fc protein to PD-L1 WT or PD-L1 4NQprotein expressed on BT549 cells at every hour time point. C. Tcell-meditated tumor cell killing (TTK) assay involving PD-L1 WT or 4NQPD-L1 protein expressed on BT549 cells. Representative phase, redfluorescent (nuclear restricted RFP), and/or green fluorescent (NucView™488 Caspase 3/7 substrate) merged images (10×) of PD-L1 WT or 4NQexpressing cells, and PD-1-expressing T cell co-cultures in the presenceof Caspase 3/7 substrate at 96 hours. T cells were activated withanti-CD3 antibody (100 ng/ml) and IL-2 (10 ng/ml). Green fluorescentcells were counted as dead cells. The quantitative ratio of dead cellsversus total cells associated with PD-L1 WT or PD-L1 4NQ protein ispresented in the bar graph at the right of the images. D. Tumor growthin BALB/c mice bearing tumors derived from injected 4T1 cells thatexpressed either PD-L1 WT or PD-L1 4NQ mutant protein. Tumor growth of4T1-luc cells was shown in vivo by bioluminescence imaging using IVIS100(at left in D). At right in D: Box plots showing the tumor volume andphotos showing tumor size in mice bearing tumors derived from 4T1 cellsexpressing PD-L1 WT versus PD-L1 4NQ proteins. Tumors were measured anddissected at the endpoint. n=8 mice per group (right). E. Intracellularcytokine staining of IFNγ in CD8+CD3+ T cell populations. Significancewas determined by two-way ANOVA, with *p<0.05 and **p<0.001; n=7 miceper group. * indicates statistically significant by Student's t test.All error bars are expressed as mean±SD of 3 independent experiments. F.Box plot showing the tumor volume in mice bearing tumors derived from4T1 cells expressing PD-L1 WT (glycosylated) proteins treated withSTM073 MAb (“73”), 100 μg, or with IgG control, 100 μg. Tumors weremeasured and dissected at the endpoint. n=7 mice per group. Treatmentwith STM073 resulted in reduced tumor volume.

FIGS. 10A-10C. Schematic Diagram of Glycosylated and Non-glycosylatedForms of the PD-L1 Protein and Western Blot Analyses. A. Schematicdepiction of wild type, glycosylated PD-L1 protein (PD-L1 WT) and fourPD-L1 protein variants, each having one glycosylated amino acid residueand three non-glycosylated amino acid residues out of the fourN-glycosylation sites of the PD-L1 protein (N35/3NQ; N192/3NQ; N200/3NQ;and N219/3NQ). The amino acids designated in black representglycosylated residues; the amino acid sites shown in red arenon-glycosylated in the variant PD-L1 proteins. B. Stable clones of BT549 expressing N35/3NQ, N192/3NQ, N200/3NQ and N219/3NQ forms of thePD-L1 protein were generated. Some of the anti-glycPD-L1 antibodiesshowed a greater level of binding to certain of the PD-L1 glycosylationvariants versus others as determined by Western blot analysis,demonstrating that those MAbs were site specific. As shown in B, forexample, MAb STM073 recognized and bound the N35/3NQ, N192/3NQ andN200/3NQ PD-L1 variants, indicating that this monoclonal antibody boundto the N35, N192 and N200 regions of glycosylated PD-L1. C. shows theresults of a Western blot of the STM073 MAb binding to lysates of livercancer cell lines.

FIG. 11. Anti-glycPD-L1 Antibodies Enhance Tumor Cell Killing by TCells. The STM073 MAb was used in different amounts in a cellularcytotoxicity assay as described in Example 10 to assess the cytotoxicityof PBMC-derived T-cells against tumor cells (BT549). FIG. 11 shows celldeath of BT549 (RFP PD-L1 (WT) cells treated with MAb STM073 in realtime. In FIG. 11, the bottom plotted line (solid orange diamonds)represents the control (no T cells from PBMCs); the solid invertedpurple triangles represent a No Antibody control; the solid greentriangles represent 10 μg/ml of the STM073 MAb used in the assay; thesolid red squares represent 20 μg/ml of the STM073 MAb used in theassay; and the solid blue circles represent 40 μg/ml of the STM073 MAbused in the assay. As observed from FIG. 11, a dose proportional killingof PD-L1 bearing tumor cells over time is demonstrated.

FIGS. 12A-12C. Binding Assays. FIGS. 12A-12C show the results of abinding assay as described in Example 10, in which the STM073 MAb (A)and the STM108 MAb (B) are seen to block binding of PD-1 to BT549 targetcells expressing WT PD-L1 in a dose dependent manner versus assaycontrols, i.e., No PD-1/Fc; No Ab; mIgG Ab controls (C).

FIG. 13. Internalization of PD-L1 by Anti-glycPD-L1 Antibodies. FIG. 13shows the results of a Western blot of PD-L1 protein from A431 cellscultured overnight in serum free medium and treated with anti-glycPD-L1antibody (10 μg) as described herein for 2 days. Shorter and longerexposures of the blot are presented. Tubulin is presented as a control.The lane designated “73,” represents treatment of A431 cells with theSTM073 MAb and shows decreased levels of PD-L1 in the STM073-treatedcells relative to treatment with control (IgG lane). The results supportthe promotion or enhancement of internalization and degradation of PD-L1by anti-glycPD-L1 antibodies, such as STM073, as described herein.

FIGS. 14A-14E. Visualization of Cellular Internalization ofAnti-glycPD-L1 Antibodies. The STM073 and STM108 MAbs were labeled andincubated with different cancer cell types and internalization wasvisualized using an IncuCyte ZOOM® instrument as described in Example11. FIG. 14A: Wild type BT 549 cells (human ductal carcinoma, breastcancer cell line) incubated with STM073; FIG. 14B: BT 549 cellsmolecularly engineered to express PD-L1 WT (glycosylated) incubated withSTM073; FIG. 14C: NCI-H226 cells (human lung cancer cell line, squamouscell mesothelioma) incubated with STM073; FIG. 14D: MCF-7 cells (humanbreast cancer cell line, adenocarcinoma) incubated with STM073; and FIG.14E: BT 549 cells expressing PD-L1 WT (glycosylated) incubated withSTM108.

FIGS. 15A-15C. Internalization and Degradation of PD-L1 FollowingBinding By Anti-glycPD-L1 Antibodies. FIGS. 15A-15C show the results oflive cell imaging of PD-L1-expressing cells incubated with dual functionanti-glycPD-L1 antibody. In FIGS. 15A-15C, the anti-PD-L1 antibody isSTM108 MAb conjugated to a red fluorescent dye, pHrodo™ Red(succinimidyl ester (pHrodo™ Red, SE), (ThermoFisher Scientific,Waltham, Mass.). pHrodo™ Red dye conjugates are non-fluorescent outsidethe cell, but fluoresce brightly red in phagosomes, which makes themuseful reagents for studies ranging from phagocytosis of bioparticles toreceptor internalization. Green staining reflects cells stained withLysoTracker® Green DND-26, which is a cell permeable green dye thatstains acidic compartments (lysosomes) in live cells imaged via livecell imaging. FIG. 15A shows that at a first time point (Time 0), STM108is internalized into cells as depicted by the intense red intracellularstaining of cells indicated by the arrow. FIG. 15B shows the weakenedintracellular red staining in the same cells depicted in FIG. 15A, at atime 2 minutes after the time point in FIG. 15A. FIG. 15C shows the lackof red intracellular staining 4 minutes after the time point in FIG.15A, which reflects the degradation of the STM108 antibody and/or theantibody-antigen complex inside the cells.

FIGS. 16A-16L. Internalization of PD-L1 Bound by Anti-PD-L1 Antibodiesin Tumor Cells Versus Total T Cells. FIGS. 16A-16L present images ofcells showing the ability of the dual function anti-glycPD-L1 antibodiesto internalize into PD-L1 positive tumor cells, but not into eitheractivated or non-activated T cells. FIGS. 16A-16D show images ofnon-activated total T cells from peripheral blood following incubationwith the following antibodies: mouse IgG antibody control (FIG. 16A);non-internalizing anti-glycPD-L1 MAb STM004 (FIG. 16B); dual functionanti-glycPD-L1 MAb STM073 (FIG. 16C); and dual function anti-glycPD-L1antibody STM108 (FIG. 16D). FIGS. 16A-16D show that none of theantibodies tested were internalized into non-activated total T cells.FIGS. 16E-16H show images of activated total T cells from peripheralblood following incubation with the following antibodies: mouse IgGantibody control (FIG. 16E); non-internalizing STM004 (FIG. 16F); dualfunction STM073 (FIG. 16G); and dual function STM108 (FIG. 16H). For Tcell activation, total T cells were mixed with beads, e.g., inert,superparamagnetic beads, covalently coupled with anti-CD3 and anti-CD28antibodies (e.g., ThermoFisher Scientific, Rochester, N.Y.) at a 1:1ratio. FIGS. 16E-16H show that virtually no internalization intoactivated total T cells was observed with any of the antibodies tested.FIGS. 16I-16L show images of NCI-H226 cells (human lung cancer cellline, squamous cell mesothelioma) following incubation with thefollowing antibodies: mouse IgG antibody control (FIG. 16I);non-internalizing anti-glycPD-L1 antibody STM004 (FIG. 16J); dualfunction anti-glycPD-L1 MAb STM073 (FIG. 16K); and dual functionanti-glycPD-L 1 MAb STM108 (FIG. 16L). FIGS. 16I-16L show that the dualfunction, internalizing STM073 and STM108 MAbs were internalized intoNCI-H226 cells following incubation with these cells, as evidenced byred intracellular staining, compared with the control antibody, mIgG(FIG. 16I) and with a non-internalizing STM004 MAb.

FIGS. 17A-17D. Anti-tumor Efficacy of an Anti-glycPD-L1 Antibody-ADC.FIGS. 17A-17D present the results of experiments evaluating the efficacyof ADCs comprising dual function anti-glycPD-L1 MAb STM108, coupled toMMAE to produce an antibody-drug conjugate (STM108-ADC) as describedherein, in killing PD-L1-expressing and non-PD-L1-expressing tumor cellsand in reducing the volume of tumors in tumor-grafted mice followinginjection of tumored animals with the STM108-ADC compared with tumoredanimals injected with controls (IgG and STM108 MAb alone). FIG. 17Ashows the % viability of PD-L1-expressing MDA-MB231 (human breastcarcinoma cell line) tumor cells (“MB231”) following exposure todifferent concentrations (nM) of STM108-ADC (filled black circles)compared with the % viability of MB231 cells molecularly engineered toknock out their expression of PD-L1 (“MB231 PDL1 KO”) following exposureto different concentrations of STM108-ADC, i.e., “ADC108” (filled blacksquares). In FIG. 17B, an MDA-MB231 mouse model of breast cancer wasused in which animals grafted with tumors derived from MB231 cells weretreated with either an IgG-MMAE control (100 μg); or with STM108-ADC(“ADC”) at 50 at 100 or at 150 μg; or with 100 μg of STM108 MAb, asindicated on the graph in FIG. 17B. Complete response (“CR”) wasobserved in 3 of 5 mice administered 100 μg STM108-ADC and in 4 of 5mice administered 150 μg STM108-ADC. FIG. 17C shows the % viability of4T1 mammary carcinoma cells molecularly engineered to express humanPD-L1 on the cell surface (“4T1 hPDL1”) following exposure to differentconcentrations (nM) of STM108-ADC (open red circles) compared with the %viability of 4T1 cells that naturally do not express PD-L1 (“4T1”)following exposure to different concentrations of STM108-ADC, i.e.,“ADC108” (open red squares). In FIG. 17D, 4T1 syngeneic mouse models ofbreast cancer were used in which the animals (Balb/c mice) were graftedwith tumors derived from 4T1 mammary carcinoma cells that had beenmolecularly engineered to express PD-L1 or the cell surface (“4T1hPD-L1”), or in which the Balb/c mice were grafted with tumors derivedfrom untransfected 4T1 mammary carcinoma cells that do not naturallyexpress PD-L1 on the cell surface (“4T1”). Animals harboring tumorsderived from the two types of 4T1 cells were treated with either anIgG-MMAE control (100 μg); or with STM108 MAb (100 μg), or withSTM108-ADC (100 m), as indicated on the graph of FIG. 17D. See, Example14.

Other aspects, features and advantages of the described embodiments willbecome apparent from the following detailed description and illustrativeexamples.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The extracellular interaction between programmed death ligand-1 protein(PD-L1) expressed on tumor cells and programmed death-1 protein (PD-1)expressed on immune effector cells, e.g., T-cells, has a marked impacton tumor-associated immune escape. Despite the clinical success ofimmune checkpoint blockade using anti-PD-1 or anti-PD-L1 antibodies, theregulatory mechanisms and structural features underlying the PD-L1 andPD-1 interaction remain largely unknown. In accordance with the findingsdescribed herein, it has been demonstrated that N-linked glycosylationof PD-L1 stabilizes the PD-L1 protein ligand as present on tumor cellsand also facilitates and enhances its binding to PD-1, which promotesthe suppression of T cell-mediated immune response. Conversely, it hasbeen found that aberrant or loss of N-linked glycosylation, such as frompartial or complete deglycosylation of the PD-L1 polypeptide expressedon tumor cells, adversely affects, e.g., weakens or disrupts, thePD-L1/PD-1 interaction and promotes the internalization and degradationof PD-L1 on the tumor cells, which, in turn, inhibits immunosuppressionand promotes effector T-cell cytotoxic activity and killing of tumorcells. In addition, because the survival of patients whose tumorsexpress highly glycosylated PD-L1 is poor, glycosylated PD-L1 isrecognized, based on the findings herein, as an effective therapeutictarget for cancer treatment. Provided and described herein are cancertherapeutics, such as dual function anti-glycPD-L1 antibodies, thatspecifically and preferentially bind and interact with glycosylatedPD-L1 to disrupt a glycosylated PD-L1/PD-1 interaction and todestabilize the PD-L1 expressed on the tumor cell surface, therebyinhibiting immunosuppression and promoting T-cell effector functionagainst the tumor cells so as to treat cancer. Tumor treatment with thedual function anti-glycosylated PD-L1 antibodies as described hereinoffers enhanced immunosuppression inhibitory effects relative toanti-PD-L1 antibodies that are not specific for glycosylated forms ofPD-L1. In embodiments, the anti-glycPD-L1 antibodies are monoclonalantibodies, designated “MAbs” herein.

Provided herein are dual function anti-glycPD-L1 antibodies that bindepitopes in the extracellular domain (ECD) of PD-L1 corresponding toglycosylation sites, particularly in the N-terminal and C-terminalportions of the PD-L1 ECD or alternatively binding so as to block ormask those glycosylation sites. The amino acids that are glycosylated inthe PD-L1 protein are in its extracellular domain, as shown in FIG. 1G,at positions 35, 192, 200 and 219 of PD-L1 as numbered in SEQ ID NO: 1herein. In accordance with the present findings, the anti-glycPD-L1antibodies described herein are dual functional in that they reduce,block, or inhibit binding of PD-L1 to PD-1 and also promote PD-L1internalization and degradation to reduce the levels of PD-L1 expressedon the tumor cell. In embodiments, the dual functional anti-glycPD-L1antibodies as described bind nonlinear, conformational epitopes. Inaddition, and without wishing to be bound by theory, the anti-glycPD-L1antibodies bind epitopes that contain glycosylated amino acids or areproximal in the three dimensional space to glycosylated amino acids;such glycosylated amino acids are believed to be particularly involvedin the PD-L1/PD-1 interaction and also involved in maintenance of thePD-L1 on the surface of the tumor cell. Not to be bound by theory,glycan structures associated with N35 within the ECD N-terminus ofglycosylated PD-L1 may comprise or contribute to a functional structureor configuration of the glycosylated PD-L1 protein that is recognizedand bound by PD-1 protein and that plays a significant role in thePD-1/PD-L1 interaction. By binding an epitope comprising the amino acidat position 35, in the N-terminal region of the PD-L1 ECD, or an epitopeproximal to position 35, the dual function anti-glycPD-L1 antibodies asdescribed mask the binding sites or regions of the glycosylated PD-L1protein that may be particularly involved in the PD-L1/PD-1 interaction.By binding or, through binding, masking, amino acids in the C-terminalregion of the PD-L1 ECD that comprises glycosylated amino acid residues(N192, N200 and N219), the dual function anti-glycPD-L1 antibodies asdescribed effectively mask the binding sites or regions of theglycosylated PD-L1 protein that may be particularly involved in thestabilization of PD-L1 on the surface of the tumor cell, therebypromoting PD-L1 internalization and degradation and reducing the levelsof PD-L1 on the tumor cells.

The dual function anti-glycPD-L1 antibodies described herein may bind toepitopes comprising or masking amino acids in both the N- and C-terminalregions of PD-L1 ECD, particularly amino acids within nonlinear,conformation epitopes, resulting in the masking or concealment by theanti-glycPD-L1 antibodies of those glycan-containing residues or regionsof the PD-L1 protein that are involved in the PD-L1/PD-1 interaction andin the stabilization of PD-L1 on the tumor cell surface. Suchanti-glycPD-L1 antibodies inhibit or block the binding of PD-L1 to PD-1and also reduce the levels of PD-L1 on the tumor cell surface such thatfewer PD-L1 molecules are available to bind to PD-1. The anti-glycPD-L1antibodies, when provided in mixtures with effector T-cells andPD-1-bearing tumor or cancer calls, promote the killing of such tumor orcancer cells by the T-cells, which are not immunosuppressed by thePD-1/PD-L1 interaction. (See, e.g., FIG. 11 and Example 9).

The Examples described herein provide experimental results showing asignificant difference, e.g., 2-3 fold, in binding of glycosylated PD-L1versus non-glycosylated PD-L1 by the anti-glycPD-L1 antibodies asdescribed herein. In embodiments, the anti-glycPD-L 1 antibodies exhibita binding affinity for glycosylated PD-L1 in the nanomolar range, e.g.,from about 5-20 nM or about 10-20 nM, relative to non-glycosylatedPD-L1. The Examples further show that the anti-glycPD-L1 antibodies areinternalized by PD-L1 expressing tumor cells but not by T cells (eitheractivated or non-activated) (FIGS. 16A-16L); the anti-glycPD-L1antibodies also reduce viability and tumor volume of PD-L1 expressingtumor cells in a mouse model harboring tumors derived from tumor celllines (FIGS. 17A-17D).

Definitions

As used herein, the term “a” or “an” may mean one or more.

As used herein, the term “or” means “and/or” unless explicitly indicatedto refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.”

As used herein, the term “another” means at least a second or more.

As used herein, the term “about” is used to indicate that a valueincludes the inherent variation of error for the device, the methodbeing employed to determine the value, or the variation that existsamong the study subjects.

As used herein, the term “programmed death ligand-1” or “PD-L1” refersto a polypeptide (the terms “polypeptide” and “protein” are usedinterchangeably herein) or any native PD-L1 from any vertebrate source,including mammals such as primates (e.g., humans, cynomolgus monkey(cyno)), dogs, and rodents (e.g., mice and rats), unless otherwiseindicated, and, in certain embodiments, included various PD-L1 isoforms,related PD-L1 polypeptides, including SNP variants thereof.

An exemplary amino acid sequence of human PD-L1 (UniProtKB/Swiss-Prot:Q9NZQ7.1; GI:83287884), is provided below:

MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEMEDKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTTTTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTHLVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET (SEQ ID NO: 1).In SEQ ID NO: 1, the amino terminal amino acids 1-18 constitute thesignal sequence of the human PD-L 1 protein. Accordingly, the maturehuman PD-L1 protein consists of amino acids 19-290 of SEQ ID NO: 1.

Abbreviations for the amino acid residues that comprise polypeptides andpeptides described herein, and conservative substitutions for theseamino acid residues are shown in Table 1 below. A polypeptide thatcontains one or more conservative amino acid substitutions or aconservatively modified variant of a polypeptide described herein refersto a polypeptide in which the original or naturally occurring aminoacids are substituted with other amino acids having similarcharacteristics, for example, similar charge,hydrophobicity/hydrophilicity, side-chain size, backbone conformation,structure and rigidity, etc. Thus, these amino acid changes cantypically be made without altering the biological activity, function, orother desired property of the polypeptide, such as its affinity or itsspecificity for antigen. In general, single amino acid substitutions innonessential regions of a polypeptide do not substantially alterbiological activity. Furthermore, substitutions of amino acids that aresimilar in structure or function are less likely to disrupt thepolypeptides' biological activity.

TABLE 1 Amino Acid Residues and Examples of Conservative Amino AcidSubstitutions Original residue Three letter code and Conservative Singleletter code substitution(s) Alanine (Ala) (A) Gly; Ser Arginine (Arg)(R) Lys; His Asparagine (Asn) (N) Gln; His Aspartic Acid (Asp) (D) Glu;Asn Cysteine (Cys) (C) Ser; Ala Glutamine (Gln) (Q) Asn Glutamic Acid(Glu) (E) Asp; Gln Glycine (Gly) (G) Ala Histidine (His) (H) Asn; GlnIsoleucine (Ile) (I) Leu; Val Leucine (Leu) (L) Ile; Val Lysine (Lys)(K) Arg; His Methionine (Met) (M) Leu; Ile; Tyr Phenylalanine (Phe) (F)Tyr; Met; Leu Proline (Pro) (P) Ala Serine (Ser) (S) Thr Threonine (Thr)(T) Ser Tryptophan (Trp) (W) Tyr; Phe Tyrosine (Tyr) (Y) Trp; Phe Valine(Val) (V) Ile; Leu

The terms “antibody,” “immunoglobulin,” and “Ig” are usedinterchangeably herein in a broad sense and specifically cover, forexample, individual anti-PD-L1 antibodies, such as the monoclonalantibodies described herein, (including agonist, antagonist,neutralizing antibodies, full length or intact monoclonal antibodies,peptide fragments of antibodies that maintain antigen binding activity),anti-unglycosylated PD-L1 antibodies and anti-glycosylated PD-L1antibodies; anti-PD-L1 antibody compositions with polyepitopic ormonoepitopic specificity, polyclonal or monovalent antibodies,multivalent antibodies, multi specific antibodies (e.g., bispecificantibodies or biparatopic antibodies, so long as they exhibit thedesired biological activity), formed from at least two intactantibodies, single chain anti-PD-L1 antibodies, and fragments ofanti-PD-L1 antibodies, as described below. An antibody can be human,humanized, chimeric and/or affinity matured. An antibody may be fromother species, for example, mouse, rat, rabbit, etc. The term “antibody”is intended to include a polypeptide product of B cells within theimmunoglobulin class of polypeptides that is able to bind to a specificmolecular antigen. An antibody is typically composed of two identicalpairs of polypeptide chains, wherein each pair has one heavy chain(about 50-70 kDa) and one light chain (about 25 kDa); and wherein theamino-terminal portion of the heavy and light chains includes a variableregion of about 100 to about 130 or more amino acids and thecarboxy-terminal portion of each chain includes a constant region (See,Antibody Engineering, Borrebaeck (ed.), 1995, Second Ed., OxfordUniversity Press.; Kuby, 1997, Immunology, Third Ed., W.H. Freeman andCompany, New York). In specific embodiments, the specific molecularantigen bound by an antibody provided herein includes a PD-L1polypeptide, a PD-L1 peptide fragment, or a PD-L1 epitope. The PD-L1polypeptide, PD-L1 peptide fragment, or PD-L1 epitope can beunglycosylated or glycosylated. In a particular embodiment, the PD-L1polypeptide, PD-L1 peptide fragment, or PD-L1 epitope is glycosylated,An antibody or a peptide fragment thereof that binds to a PD-L1 antigencan be identified, for example, by immunoassays, Biacore, or othertechniques known to those of skill in the art. An antibody or a fragmentthereof binds specifically to a PD-L1 antigen when it binds to a PD-L1antigen with higher affinity than to any cross-reactive antigen asdetermined using experimental techniques, such as radioimmunoassays (MA)and enzyme linked immunosorbent assays (ELISAs). Typically, a specificor selective binding reaction will be at least twice background signalor noise, and more typically more than 5-10 times background signal ornoise. See, e.g., Fundamental Immunology Second Edition, Paul, ed.,1989, Raven Press, New York at pages 332-336 for a discussion regardingantibody specificity.

Antibodies provided herein include, but are not limited to, syntheticantibodies, monoclonal antibodies, recombinantly produced antibodies,multispecific antibodies (including bi-specific antibodies), humanantibodies, humanized antibodies, camelized antibodies, chimericantibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, andfunctional fragments (e.g., antigen-binding fragments such as PD-L1binding fragments) of any of the above. A binding fragment refers to aportion of an antibody heavy or light chain polypeptide, such as apeptide portion, that retains some or all of the binding activity of theantibody from which the fragment is derived. Non-limiting examples offunctional fragments (e.g., antigen-binding fragments such as PD-L1binding fragments) include single-chain Fvs (scFv) (e.g., includingmonospecific, bispecific, biparatopic, monovalent (e.g., with a singleV_(H) or V_(L) domain) or bivalent, etc.), Fab fragments, F(ab′)fragments, F(ab)₂ fragments, F(ab′)₂ fragments, disulfide-linked Fvs(sdFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodiesand minibodies. In particular, antibodies provided herein includeimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, for example, antigen binding domains ormolecules that contain an antigen-binding site that binds to a PD-L1antigen, in particular, a glycosylated PD-L1 antigen, (e.g., one or morecomplementarity determining regions (CDRs) of an anti-PD-L1 antibody).Description of such antibody fragments can be found in, for example,Harlow and Lane, 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York; Molec. Biology and Biotechnology: AComprehensive Desk Reference, Myers (ed.), New York: VCH Publisher,Inc.; Huston et al., 1993, Cell Biophysics, 22:189-224; Plückthun andSkerra, 1989, Meth. Enzymol., 178:497-515 and in Day, E. D., 1990,Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y.The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulinmolecule. In certain embodiments, the anti-PD-L1 antibodies are fullyhuman, such as fully human monoclonal anti-PD-L1 antibodies. In certainembodiments, the anti-PD-L1 antibodies are humanized, such as humanizedmonoclonal anti-PD-L1 antibodies. In certain embodiments, the antibodiesprovided herein are IgG antibodies, or a class (e.g., human IgG1 orIgG4) or subclass thereof, in particular, IgG1 subclass antibodies.

A four-chain antibody unit is a heterotetrameric glycoprotein composedof two identical light (L) chains and two identical heavy (H) chains. Inthe case of IgGs, the molecular weight of the four-chain (unreduced)antibody unit is generally about 150,000 daltons. Each L chain is linkedto a H chain by one covalent disulfide bond, while the two H chains arelinked to each other by one or more disulfide bonds depending on the Hchain isotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. At the N-terminus, each H chain has a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its carboxy terminus. The V_(L) domain is aligned withthe V_(H) domain, and the C_(L) domain is aligned with the firstconstant domain of the heavy chain (C_(H1)). Particular amino acidresidues are believed to form an interface between the light chain andheavy chain variable domains. The pairing of a V_(H) and V_(L) togetherforms a single antigen-binding site (although certain V_(H) and V_(L)domains can bind antigen independently of the counterpart V_(H) orV_(L), respectively). The basic structure of immunoglobulin molecules isunderstood by those having skill in the art. For example, the structureand properties of the different classes of antibodies may be found inStites, Daniel P. et al., 1994, Basic and Clinical Immunology, 8thedition, Appleton & Lange, Norwalk, Conn., page 71 and Chapter 6.

As used herein, the term “antigen” or “target antigen” is apredetermined molecule to which an antibody can selectively bind. Atarget antigen can be a polypeptide, peptide, carbohydrate, nucleicacid, lipid, hapten, or other naturally occurring or synthetic compound.In embodiments, a target antigen is a small molecule. In certainembodiments, the target antigen is a polypeptide or peptide, preferablya glycosylated PD-L1 polypeptide.

As used herein, the term “antigen binding fragment,” “antigen bindingdomain,” “antigen binding region,” and similar terms refer to thatportion of an antibody which includes the amino acid residues thatinteract with an antigen and confer on the antibody as binding agent itsspecificity and affinity for the antigen (e.g., the CDRs of an antibodyare antigen binding regions). The antigen binding region can be derivedfrom any animal species, such as rodents (e.g., rabbit, rat, or hamster)and humans. In specific embodiments, the antigen binding region can beof human origin.

An “isolated” antibody is substantially free of cellular material orother contaminating proteins from the cell or tissue source and/or othercontaminant components from which the antibody is derived, or issubstantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of an antibody in which the antibody isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, an antibody that issubstantially free of cellular material includes preparations of anantibody that have less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1%(by dry weight) of heterologous protein (also referred to herein as a“contaminating protein”). In certain embodiments, when the antibody isrecombinantly produced, it is substantially free of culture medium,e.g., culture medium represents less than about 20%, 15%, 10%, 5%, or 1%of the volume of the protein preparation. In certain embodiments, whenthe antibody is produced by chemical synthesis, it is substantially freeof chemical precursors or other chemicals, for example, it is separatedfrom chemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the antibodyhave less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight)of chemical precursors or compounds other than the antibody of interest.Contaminant components can also include, but are not limited to,materials that would interfere with therapeutic uses for the antibody,and can include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the antibody ispurified (1) to greater than or equal to 95% by weight of the antibody,as determined by the Lowry method (Lowry et al., 1951, J. Bio. Chem.,193: 265-275), such as 95%, 96%, 97%, 98%, or 99%, by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or silver stain. Isolated antibody also includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present. Anisolated antibody is typically prepared by at least one purificationstep. In some embodiments, the antibodies provided herein are isolated.

As used herein, the term “binds” or “binding” refers to an interactionbetween molecules including, for example, to form a complex.Illustratively, such interactions embrace non-covalent interactions,including hydrogen bonds, ionic bonds, hydrophobic interactions, and/orvan der Waals interactions. A complex can also include the binding oftwo or more molecules held together by covalent or non-covalent bonds,interactions, or forces. The strength of the total non-covalentinteractions between a single antigen-binding site of an antibody andits epitope on a target (antigen) molecule, such as PD-L1, is theaffinity of the antibody or functional fragment for that epitope. Theratio of association (k_(on)) to dissociation (k_(off)) of an antibodyto a monovalent antigen (k_(on)/k_(off)) is the association constantK_(a), which is a measure of affinity. The value of K_(a) varies fordifferent complexes of antibody and antigen and depends on both k_(on)and k_(off). The association constant K_(a) for an antibody providedherein may be determined using any method provided herein or any othermethod known to those skilled in the art. The affinity at one bindingsite does not always reflect the true strength of the interactionbetween an antibody and an antigen. When complex antigens containingmultiple antigenic determinants, such as a glycosylated PD-L1, come intocontact with antibodies containing multiple binding sites, theinteraction of antibody with antigen at one site will increase theprobability of an interaction at a second binding site. The strength ofsuch multiple interactions between a multivalent antibody and antigen iscalled the avidity. The avidity of an antibody can be a better measureof its binding capacity than is the affinity of its individual bindingsites. For example, high avidity can compensate for low affinity as issometimes found for pentameric IgM antibodies, which can have a loweraffinity than IgG, but the high avidity of IgM, resulting from itsmultivalence, enables it to bind antigen effectively.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., a binding protein such as an antibody) and its binding partner(e.g., an antigen). Unless indicated otherwise, as used herein, “bindingaffinity” refers to intrinsic binding affinity which reflects a 1:1interaction between members of a binding pair (e.g., antibody andantigen or receptor and ligand). The affinity of a binding molecule Xfor its binding partner Y can generally be represented by thedissociation constant (K_(d)). Affinity can be measured by commonmethods known in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,while high-affinity antibodies generally bind antigen faster and tend toremain bound longer to antigen. A variety of methods for measuringbinding affinity are known in the art, any of which may be used forpurposes of the present disclosure. Specific illustrative embodimentsinclude the following: In one embodiment, the “K_(d)” or “K_(d) value”is measured by assays known in the art, for example, by a binding assay.The K_(d) can be measured in a radiolabeled antigen binding assay (RIA),for example, performed with the Fab portion of an antibody of interestand its antigen (Chen et al., 1999, J. Mol. Biol., 293:865-881). TheK_(d) or K_(d) value may also be measured by using surface plasmonresonance (SPR) assays (by Biacore) using, for example, a BIAcore™-2000or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.), or by biolayerinterferometry (BLI) using, for example, the OctetQK384 system(ForteBio, Menlo Park, Calif.), or by quartz crystal microbalance (QCM)technology. An “on-rate” or “rate of association” or “association rate”or “k_(on)” can also be determined with the same surface plasmonresonance or biolayer interferometry techniques described above, using,for example, a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc.,Piscataway, N.J.), or the OctetQK384 system (ForteBio, Menlo Park,Calif.).

The terms “anti-PD-L1 antibody,” “an antibody that specifically binds toPD-L1,” or “antibody that is specific for PD-L1,” “antibodies thatspecifically bind to a PD-L1 epitope,” “an antibody that selectivelybinds to PD-L1,” “antibodies that selectively bind to a PD-L1 epitope,”“an antibody that preferentially binds to PD-L1, and analogous terms areused interchangeably herein and refer to antibodies capable of bindingPD-L1, i.e., glycosylated or WT PD-L1, with sufficient affinity andspecificity, particularly compared with non-glycosylated PD-L1 orglycosylation mutants of PD-L1. “Preferential binding” of theanti-glycPD-L1 antibodies as provided herein may be determined ordefined based on the quantification of fluorescence intensity of theantibodies' binding to PD-L1, i.e., glycosylated PD-L1 polypeptide, orPD-L1 WT, or glycosylated PD-L1 expressed on cells versus an appropriatecontrol, such as binding to non-glycosylated or variant PD-L1 (e.g., 4NQPD-L1), or to cells expressing a non-glycosylated or variant form ofPD-L1 (e.g., 4NQ PD-L1), for example, molecularly engineered cells, celllines or tumor cell isolates, such as described herein, e.g., in Example6. Preferential binding of an anti-glycPD-L1 antibody as described to aglycosylated PD-L1 polypeptide or to a glycosylated PD-L1 (PD-L1WT)-expressing cell is indicated by a measured fluorescent bindingintensity (MFI) value, as assessed by cell flow cytometry, of at least1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least10-fold, at least 15-fold, at least 20-fold or greater, as compared withbinding of the antibody to a non-glycosylated or mutant glycosylatedPD-L1 polypeptide or a non-glycosylated or mutant glycosylatedPD-L1-expressing cell, and wherein the antibody to be assayed isdirectly or indirectly detectable by a fluorescent label or marker. Inan embodiment, the antibody is directly labeled with a fluorescentmarker, such as FITC. In embodiments, an anti-glycPD-L1 antibody thatpreferentially or selectively binds glycosylated PD-L1 exhibits an MFIvalue of from 1.5-fold to 25-fold, or from 2-fold to 20-fold, or from3-fold to 15-fold, or from 4-fold to 8-fold, or from 2-fold to 10-fold,or from 2-fold to 5-fold or more greater than the MFI value of the sameantibody for binding a non-glycosylated PD-L1 or a PD-L1 glycosylationvariant as described herein e.g., 4NQ PD-L1, which is not glycosylated.Fold-fluorescence intensity values between and equal to all of theforegoing are intended to be included. In an embodiment, theanti-glycPD-L1 antibodies specifically and preferentially bind to aglycosylated PD-L1 polypeptide, such as a glycosylated PD-L1 antigen,peptide fragment, or epitope (e.g., human glycosylated PD-L1 such as ahuman glycosylated PD-L1 polypeptide, antigen or epitope). An antibodythat specifically binds to PD-L1, (e.g., glycosylated or wild type humanPD-L1) can bind to the extracellular domain (ECD) or a peptide derivedfrom the ECD of PD-L1. An antibody that specifically binds to a PD-L1antigen (e.g., human PD-L1) can be cross-reactive with related antigens(e.g., cynomolgus (cyno) PD-L1). In a preferred embodiment, an antibodythat specifically binds to a PD-L1 antigen does not cross-react withother antigens. An antibody that specifically binds to a PD-L1 antigencan be identified, for example, by immunofluorescence binding assays,immunoassay methods, immunohistochemistry assay methods, Biacore, orother techniques known to those of skill in the art.

In certain other embodiments, an antibody that binds to PD-L1, asdescribed herein, has a dissociation constant (K_(d)) of less than orequal to 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM,11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 0.9 nM, 0.8 nM, 0.7nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM, and/or is greaterthan or equal to 0.1 nM. In certain embodiments, an anti-PD-L1 antibodybinds to an epitope of PD-L1 that is conserved among PD-L1 proteins fromdifferent species (e.g., between human and cynomolgus PD-L1). Anantibody binds specifically to a PD-L1 antigen when it binds to a PD-L1antigen with higher affinity than to any cross reactive antigen asdetermined using experimental techniques, such as radioimmunoassays (MA)and enzyme linked immunosorbent assays (ELISAs). Typically a specific orselective reaction will be at least twice background signal or noise andcan be more than 10 times background. See, e.g., Fundamental ImmunologySecond Edition, Paul, ed., 1989, Raven Press, New York at pages 332 336for a discussion regarding antibody specificity. In such embodiments,the extent of binding of the antibody to a “non-target” protein will beless than about 10% of the binding of the antibody to its particulartarget protein, for example, as determined by fluorescence activatedcell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

Anti-PD-L1 antibodies as described herein include anti-glycosylatedPD-L1 antibodies or anti-wild type PD-L1 antibodies, wherein wild typePD-L1 protein is glycosylated, which are specific for glycosylatedPD-L1. In some embodiments, the anti-glycosylated PD-L1 antibodies bindto a linear glycosylation motif of PD-L1. In some embodiments, theanti-glycosylated PD-L1 antibodies bind to a peptide sequence that islocated near one or more of the glycosylation motifs in threedimensions. In some embodiments, the anti-glycosylated PD-L1 antibodiesselectively bind to one or more glycosylation motifs of PD-L1 or a PD-L1peptide having a glycosylation motif of PD-L1 relative to unglycosylatedPD-L1. In other embodiments, the anti-glycPD-L1 antibodies bind to alinear epitope comprising amino acids of the PD-L1 protein. In someembodiments, the anti-glycosylated PD-L1 antibodies selectively bind toone or more glycosylation motifs of PD-L1, in which the glycosylationmotifs comprise N35, N192 N200, and/or N219 of the PD-L1 polypeptide ofSEQ ID NO: 1. In yet other embodiments, the anti-glycPD-L1 antibodiesbind to a conformational (nonlinear) epitope comprising amino acids ofthe PD-L1 protein. In some embodiments, an anti-glycPD-L1 antibody, or abinding portion thereof, binds to glycosylated PD-L1 with a K_(d) lessthan at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or 90% of the K_(d) exhibited relative to unglycosylated PD-L1. Incertain embodiments, an anti-glycPD-L1 antibody, or a binding portionthereof, binds to glycosylated PD-L1 with a K_(d) less than 50% of theK_(d) exhibited relative to unglycosylated PD-L1. In some embodiments,an anti-glycPD-L1 antibody, or a binding portion thereof, binds toglycosylated PD-L1 with a K_(d) that is less than 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, or 90% of the K_(d) exhibited relative to unglycosylatedPD-L1. In further aspects, an anti-glycPD-L1 antibody, or a bindingportion thereof, binds to glycosylated PD-L1 with a K_(d) at least 5-10times less than the K_(d) exhibited relative to unglycosylated PD-L1. Inan embodiment, an anti-glycPD-L1 antibody, or a binding portion thereof,binds to glycosylated PD-L1 with a K_(d) at least 10 times less than theK_(d) exhibited relative to unglycosylated PD-L1. In certainembodiments, the antibody binds to glycosylated PD-L1 with a K_(d) thatis no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% ofthe K_(d) exhibited by binding to unglycosylated PD-L1. In anembodiment, an anti-glycPD-L1 antibody, or a binding portion thereof,binds to glycosylated PD-L1 with a nanomolar affinity, such as anaffinity of from 5-20 nM or from 10-20 nM, inclusive of the lower andupper values.

In an embodiment, in a cell flow cytometry binding assay as described inExample 6, the antibody exhibits binding as expressed as MFI to cellsexpressing WT PD-L1 that is at least or is 1.5 times, 2 times, 3, times,4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times greaterthan the MFI for binding to cells expressing unglycosylated PD-L1, andin certain embodiments, is no more than 10 times, 20 times 50 times or100 times greater than the MFI for binding to cells expressingunglycosylated PD-L1.

As used herein in reference to an antibody, the term “heavy (H) chain”refers to a polypeptide chain of about 50-70 kDa, wherein theamino-terminal portion includes a variable (V) region (also called Vdomain) of about 115 to 130 or more amino acids and a carboxy-terminalportion that includes a constant (C) region. The constant region (orconstant domain) can be one of five distinct types, (e.g., isotypes)referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ),based on the amino acid sequence of the heavy chain constant region. Thedistinct heavy chains differ in size: α, δ and γ contain approximately450 amino acids, while μ and ε contain approximately 550 amino acids.When combined with a light chain, these distinct types of heavy chainsgive rise to five well known classes (e.g., isotypes) of antibodies,namely, IgA, IgD, IgE, IgG and IgM, respectively, including foursubclasses of IgG, namely IgG1, IgG2, IgG3 and IgG4. An antibody heavychain can be a human antibody heavy chain.

As used herein in reference to an antibody, the term “light (L) chain”refers to a polypeptide chain of about 25 kDa, wherein theamino-terminal portion includes a variable domain of about 100 to about110 or more amino acids and a carboxy-terminal portion that includes aconstant region. The approximate length of a light chain (both the V andC domains) is 211 to 217 amino acids. There are two distinct types oflight chains, referred to as kappa (κ) and lambda (λ), based on theamino acid sequence of the constant domains. Light chain amino acidsequences are well known in the art. An antibody light chain can be ahuman antibody light chain.

As used herein, the term “variable (V) region” or “variable (V) domain”refers to a portion of the light (L) or heavy (H) chains of an antibodypolypeptide that is generally located at the amino-terminus of the L orH chain. The H chain V domain has a length of about 115 to 130 aminoacids, while the L chain V domain is about 100 to 110 amino acids inlength. The H and L chain V domains are used in the binding andspecificity of each particular antibody for its particular antigen. TheV domain of the H chain can be referred to as “V_(H).” The V region ofthe L chain can be referred to as “V_(L).” The term “variable” refers tothe fact that certain segments of the V domains differ extensively insequence among different antibodies. While the V domain mediates antigenbinding and defines specificity of a particular antibody for itsparticular antigen, the variability is not evenly distributed across the110-amino acid span of antibody V domains. Instead, the V domainsconsist of less variable (e.g., relatively invariant) stretches calledframework regions (FRs) of about 15-30 amino acids separated by shorterregions of greater variability (e.g., extreme variability) called“hypervariable regions” or “complementarity determining regions” (CDRs)that are each about 9-12 amino acids long. The V domains of antibody Hand L chains each comprise four FRs, largely adopting a β sheetconfiguration, connected by three hypervariable regions, called, whichform loops connecting, and in some cases forming part of, the β sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see, e.g., Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md.)). The C domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as antibody dependent cellular cytotoxicity(ADCC) and complement dependent cytotoxicity (CDC). The V domains differextensively in sequence among different antibody classes or types. Thevariability in sequence is concentrated in the CDRs, which are primarilyresponsible for the interaction of the antibody with antigen. Inspecific embodiments, the variable domain of an antibody is a human orhumanized variable domain.

As used herein, the terms “complementarity determining region,” “CDR,”“hypervariable region,” “HVR,” and “HV” are used interchangeably. A“CDR” refers to one of three hypervariable regions (H1, H2 or H3) withinthe non-framework region of the antibody V_(H) β-sheet framework, or toone of three hypervariable regions (L1, L2 or L3) within thenon-framework region of the antibody V_(L) β-sheet framework. The term,when used herein, refers to the regions of an antibody V domain that arehypervariable in sequence and/or form structurally defined loops.Generally, antibodies comprise six hypervariable regions: three (H1, H2,H3) in the V_(H) domain and three (L1, L2, L3) in the V_(L) domain.Accordingly, CDRs are typically highly variable sequences interspersedwithin the framework region sequences of the V domain. “Framework” or“FR” residues are those variable region residues flanking the CDRs. FRresidues are present, for example, in chimeric, humanized, human, domainantibodies, diabodies, linear antibodies, bispecific, or biparatopicantibodies.

A number of hypervariable region delineations are in use and areencompassed herein. CDR regions are well known to those skilled in theart and have been defined by, for example, Kabat as the regions of mosthypervariability within the antibody V domains (Kabat et al., 1977, J.Biol. Chem., 252:6609-6616; Kabat, 1978, Adv. Prot. Chem., 32:1-75). TheKabat CDRs are based on sequence variability and are the most commonlyused (see, e.g., Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md.). CDR region sequences also havebeen defined structurally by Chothia as those residues that are not partof the conserved β-sheet framework, and thus are able to adopt differentconformations (Chothia et al., 1987, J. Mol. Biol., 196:901-917).Chothia refers instead to the location of the structural loops. The endof the Chothia CDR-H1 loop when numbered using the Kabat CDR numberingconvention varies between H32 and H34 depending on the length of theloop (this is because the Kabat numbering scheme places the insertionsat H35A and H35B; if neither 35A nor 35B is present, the loop ends at32; if only 35A is present, the loop ends at 33; if both 35A and 35B arepresent, the loop ends at 34). Both numbering systems and terminologiesare well recognized in the art.

Recently, a universal numbering system has been developed and widelyadopted, ImMunoGeneTics (IMGT) Information System® (Lefranc et al.,2003, Dev. Comp. Immunol., 27(1):55-77). IMGT is an integratedinformation system specializing in immunoglobulins (Ig), T cellreceptors (TR) and the major histocompatibility complex (MHC) of humanand other vertebrates. Herein, the CDRs are referred to in terms of boththe amino acid sequence and the location within the light or heavychain. As the “location” of the CDRs within the structure of theimmunoglobulin V domain is conserved between species and present instructures called loops, by using numbering systems that align variabledomain sequences according to structural features, CDR and frameworkresidues and are readily identified. This information can be used ingrafting and in the replacement of CDR residues from immunoglobulins ofone species into an acceptor framework from, typically, a humanantibody. An additional numbering system (AHon) has been developed byHonegger et al., 2001, J. Mol. Biol. 309:657-670. Correspondence betweenthe numbering system, including, for example, the Kabat numbering andthe IMGT unique numbering system, is well known to one skilled in theart (see, e.g., Kabat, Id.; Chothia et al., 1987, J. Mol. Biol.,196:901-917, supra; Martin, 2010, Antibody Engineering, Vol. 2, Chapter3, Springer Verlag; and Lefranc et al., 1999, Nuc. Acids Res.,27:209-212).

CDR region sequences have also been defined by AbM, Contact and IMGT.The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular'sAbM antibody modeling software (see, e.g., Martin, Id.). The “contact”hypervariable regions are based on an analysis of the available complexcrystal structures. The residues from each of these hypervariableregions or CDRs are noted below.

Exemplary delineations of CDR region sequences are illustrated in Table2 below. The positions of CDRs within a canonical antibody variableregion have been determined by comparison of numerous structures(Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Morea et al.,2000, Methods, 20:267-279). Because the number of residues within ahypervariable region varies in different antibodies, additional residuesrelative to the canonical positions are conventionally numbered with a,b, c and so forth next to the residue number in the canonical variableregion numbering scheme (Al-Lazikani et al., Id.). Such nomenclature issimilarly well known to those skilled in the art.

TABLE 2 Exemplary Delineations of CDR Region Sequences IMGT Kabat AbMChothia Contact V_(H) CDR1 27-38 31-35 26-35 26-32 30-35 V_(H) CDR256-65 50-65 50-58 53-55 47-58 V_(H) CDR3 105-117  95-102  95-102  96-101 93-101 V_(L) CDR1 27-38 24-34 24-34 26-32 30-36 V_(L) CDR2 56-65 50-5650-56 50-52 46-55 V_(L) CDR3 105-117 89-97 89-97 91-96 89-96

An “affinity matured” antibody is one with one or more alterations(e.g., amino acid sequence variations, including changes, additionsand/or deletions) in one or more HVRs thereof that result in animprovement in the affinity of the antibody for antigen, compared to aparent antibody which does not possess those alteration(s). In certainembodiments, affinity matured antibodies will have nanomolar or evenpicomolar affinities for the target antigen, such as the glycosylatedPD-L1. Affinity matured antibodies are produced by procedures known inthe art. For reviews, see Hudson and Souriau, 2003, Nature Medicine9:129-134; Hoogenboom, 2005, Nature Biotechnol., 23:1105-1116; Quirozand Sinclair, 2010, Revista Ingeneria Biomedia 4:39-51.

A “chimeric” antibody is one in which a portion of the H and/or L chain,e.g., the V domain, is identical with or homologous to a correspondingamino acid sequence in an antibody derived from a particular species orbelonging to a particular antibody class or subclass, while theremainder of the chain(s), e.g., the C domain, is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well as afragment of such an antibody, so long as it exhibits the desiredbiological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison etal., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855).

A “humanized” nonhuman (e.g., murine) antibody is a chimeric form of anantibody that refers to a human immunoglobulin sequence (e.g., recipientantibody) in which the native CDR residues are replaced by residues fromthe corresponding CDRs of a nonhuman species (e.g., donor antibody) suchas mouse, rat, rabbit or nonhuman primate having the desiredspecificity, affinity, and capacity for antigen binding and interaction.In some instances, one or more FR region residues of the humanimmunoglobulin may also be replaced by corresponding nonhuman residues.In addition, humanized antibodies can comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine the humanized antibody'sperformance. One or more modifications in the CDR regions can also bemade to improve and refine the humanized antibody's performance, forexample, in an affinity matured antibody. A humanized antibody H or Lchain may comprise substantially all of at least one or more variableregions, in which all or substantially all of the CDRs correspond tothose of a nonhuman immunoglobulin and all or substantially all of theFRs are those of a human immunoglobulin sequence. In certainembodiments, the humanized antibody will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. While known to those skilled in the art, further detailsmay be found, if desired, in, e.g., Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-329; and Presta,1992, Curr. Op. Struct. Biol., 2:593-596; Carter et al., 1992, Proc.Natl. Acd. Sci. USA, 89:4285-4289; and U.S. Pat. No. 6,800,738 (issuedOct. 5, 2004), U.S. Pat. No. 6,719,971 (issued Sep. 27, 2005), U.S. Pat.No. 6,639,055 (issued Oct. 28, 2003), U.S. Pat. No. 6,407,213 (issuedJun. 18, 2002), and U.S. Pat. No. 6,054,297 (issued Apr. 25, 2000).

The terms “human antibody” and “fully human antibody” are usedinterchangeably herein and refer to an antibody that possesses an aminoacid sequence which corresponds to that of an antibody produced by ahuman and/or has been made using any of the techniques for making humanantibodies as practiced by those skilled in the art. This definition ofa human antibody specifically excludes a humanized antibody comprisingnon-human antigen-binding residues. Human antibodies can be producedusing various techniques known in the art, including phage-displaylibraries (Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks etal., 1991, J. Mol. Biol., 222:581) and yeast display libraries (Chao etal., 2006, Nature Protocols, 1: 755-768). Also available for thepreparation of human monoclonal antibodies are methods described in Coleet al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77; Boerner et al., 1991, J. Immunol., 147(1):86-95. See also van Dijkand van de Winkel, 2001, Curr. Opin. Pharmacol., 5:368-74. Humanantibodies can be prepared by administering an antigen to a transgenicanimal whose endogenous Ig loci have been disabled, e.g, a mouse, andthat has been genetically modified to harbor human immunoglobulin geneswhich encode human antibodies, such that human antibodies are generatedin response to antigenic challenge (see, e.g., Jakobovits, A., 1995,Curr. Opin. Biotechnol., 6(5):561-6; Brüggemann and Taussing, 1997,Curr. Opin. Biotechnol., 8(4):455-8; and U.S. Pat. Nos. 6,075,181 and6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li etal., 2006, Proc. Natl. Acad. Sci. USA, 103:3557-3562 regarding humanantibodies generated via a human B-cell hybridoma technology. Inspecific embodiments, human antibodies comprise a variable region andconstant region of human origin. “Fully human” anti-PD-L1 antibodies, incertain embodiments, can also encompass antibodies which bind PD-L1polypeptides and are encoded by nucleotide sequences that are naturallyoccurring somatic variants of human germline immunoglobulin nucleic acidsequence. In a specific embodiment, the anti-PD-L1 antibodies providedherein are fully human antibodies. The term “fully human antibody”includes antibodies having variable and constant regions correspondingto human germline immunoglobulin sequences as described by Kabat et al.(See Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). The phrase “recombinant human antibody”includes human antibodies that are prepared, expressed, created, orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell; antibodiesisolated from a recombinant, combinatorial human antibody library;antibodies isolated from an animal (e.g., a mouse or cow) that istransgenic and/or transchromosomal for human immunoglobulin genes (seee.g., Taylor, L. D. et al., 1992, Nucl. Acids Res. 20:6287-6295); orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies can have variable andconstant regions derived from human germline immunoglobulin sequences(See Kabat, E. A. et al., Id). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

As used herein, the term “epitope” is the site(s) or region(s) on thesurface of an antigen molecule to which a single antibody moleculebinds, such as a localized region on the surface of an antigen, e.g., aPD-L1 polypeptide or a glycosylated PD-L1 polypeptide that is capable ofbeing bound by one or more antigen binding regions of an anti-PD-L1 oranti-glycPD-L1 antibody. An epitope can be immunogenic and capable ofeliciting an immune response in an animal. Epitopes need not necessarilybe immunogenic. Epitopes often consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains and havespecific three dimensional structural characteristics as well asspecific charge characteristics. An epitope can be a linear epitope or aconformational epitope. A region of a polypeptide contributing to anepitope can be contiguous amino acids of the polypeptide, forming alinear epitope, or the epitope can be formed from two or morenon-contiguous amino acids or regions of the polypeptide, typicallycalled a conformational epitope. The epitope may or may not be athree-dimensional surface feature of the antigen. In certainembodiments, a PD-L1 epitope is a three-dimensional surface feature of aPD-L1 polypeptide. In other embodiments, a PD-L1 epitope is linearfeature of a PD-L1 polypeptide. In some embodiments, the PD-L1 epitopeis glycosylated at one or more sites. Generally an antigen has severalor many different epitopes and can react with many different antibodies.In a particular embodiment, an anti-glycPD-L1 antibody as describedherein binds an epitope of PD-L1, especially glycosylated PD-L1, that isa conformational epitope.

An antibody binds “an epitope” or “essentially the same epitope” or “thesame epitope” as a reference antibody, when the two antibodies recognizeidentical, overlapping, or adjacent epitopes in a three-dimensionalspace. The most widely used and rapid methods for determining whethertwo antibodies bind to identical, overlapping, or adjacent epitopes in athree-dimensional space are competition assays, which can be configuredin a number of different formats, for example, using either labeledantigen or labeled antibody. In some assays, the antigen is immobilizedon a 96-well plate, or expressed on a cell surface, and the ability ofunlabeled antibodies to block the binding of labeled antibodies toantigen is measured using a detectable signal, e.g., radioactive,fluorescent or enzyme labels. In addition, the epitope of the antibodycan be determined and then compared using methods known in the art and,for example, described in Example 8 herein.

The term “compete” when used in the context of anti-PD-L1 antibodiesthat compete for the same epitope or binding site on a PD-L1 targetprotein or peptide thereof means competition as determined by an assayin which the antibody under study prevents, blocks, or inhibits thespecific binding of a reference molecule (e.g., a reference ligand, orreference antigen binding protein, such as a reference antibody) to acommon antigen (e.g., PD-L1 or a fragment thereof). Numerous types ofcompetitive binding assays can be used to determine if a test antibodycompetes with a reference antibody for binding to PD-L1 (e.g., humanPD-L1 or human glycosylated PD-L1). Examples of assays that can beemployed include solid phase direct or indirect radioimmunoassay (RIA);solid phase direct or indirect enzyme immunoassay (EIA); sandwichcompetition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland etal., 1986, J. Immunol. 137:3614-3619); solid phase direct labeled assay;solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane,1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solidphase direct label RIA using labeled iodine (1¹²⁵ label) (see, e.g.,Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase directbiotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand.J. Immunol. 32:77-82). Typically, such an assay involves the use of apurified antigen (e.g., PD-L1 such as human PD-L1 or glycosylated PD-L1)bound to a solid surface, or cells bearing either of an unlabeled testantigen binding protein (e.g., test anti-PD-L1 antibody) or a labeledreference antigen binding protein (e.g., reference anti-PD-L1 antibody).Competitive inhibition can be measured by determining the amount oflabel bound to the solid surface or cells in the presence of a knownamount of the test antigen binding protein. Usually the test antigenbinding protein is present in excess. Antibodies identified bycompetition assay (competing antibodies) include antibodies binding tothe same epitope as the reference antibody and/or antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference antibody causing steric hindrance to occur. Additional detailsregarding methods for determining competitive binding are describedherein. Usually, when a competing antibody protein is present in excess,it will inhibit specific binding of a reference antibody to a commonantigen by at least 15%, or at least 23%, for example, withoutlimitation, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% or greater, as wellas percent amounts between the amounts stated. In some instance, bindingis inhibited by at least 80%, 85%, 90%, 95%, 96% or 97%, 98%, 99% ormore.

As used herein, the term “blocking” antibody or an “antagonist” antibodyrefers to an antibody that prevents, inhibits, blocks, or reducesbiological or functional activity of the antigen to which it binds.Blocking antibodies or antagonist antibodies can substantially orcompletely prevent, inhibit, block, or reduce the biological activity orfunction of the antigen. For example, a blocking anti-PD-L1 antibody canprevent, inhibit, block, or reduce the binding interaction between PD-L1and PD-1, thus preventing, blocking, inhibiting, or reducing theimmunosuppressive functions associated with the PD-1/PD-L1 interaction.The terms block, inhibit, and neutralize are used interchangeably hereinand refer to the ability of anti-PD-L1 antibodies as described herein toprevent or otherwise disrupt or reduce the PD-L1/PD-1 interaction.

As used herein, the term “polypeptide” or “peptide” refers to a polymerof amino acids of three or more amino acids in a serial array, linkedthrough peptide bonds. “Polypeptides” can be proteins, proteinfragments, protein analogs, oligopeptides and the like. The amino acidsthat comprise the polypeptide may be naturally derived or synthetic. Thepolypeptide may be purified from a biological sample. For example, aPD-L1 polypeptide or peptide may be composed of at least 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25contiguous amino acids of human PD-L1 or glycosylated PD-L1. In someembodiments, the polypeptide has at least 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, or 285 contiguous amino acids of human PD-L1 or glycosylatedPD-L1. In certain embodiments, the PD-L1 polypeptide comprises at least5 contiguous amino acid residues, at least 10 contiguous amino acidresidues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least 80 contiguous aminoacid residues, at least 90 contiguous amino acid residues, at least 100contiguous amino acid residues, at least 125 contiguous amino acidresidues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acidresidues, at least 250 contiguous amino acid residues of the amino acidsequence of a PD-L1 polypeptide or a glycosylated PD-L1 polypeptide.

As used herein, the term “analog” refers to a polypeptide that possessesa similar or identical function as a reference polypeptide but does notnecessarily comprise a similar or identical amino acid sequence of thereference polypeptide, or possess a similar or identical structure ofthe reference polypeptide. The reference polypeptide may be a PD-L1polypeptide, a fragment of a PD-L1 polypeptide, an anti-PD-L1 antibody,or an anti-glycPD-L1 antibody. A polypeptide that has a similar aminoacid sequence with a reference polypeptide refers to a polypeptidehaving an amino acid sequence that is at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical to the amino acidsequence of the reference polypeptide, which can be a PD-L1 polypeptideor an anti-PD-L1 antibody as described herein. A polypeptide withsimilar structure to a reference polypeptide refers to a polypeptidethat has a secondary, tertiary or quaternary structure similar to thatof the reference polypeptide, which can be a PD-L1 polypeptide or aPD-L1 antibody described herein. The structure of a polypeptide candetermined by methods known to those skilled in the art, including butnot limited to, X-ray crystallography, nuclear magnetic resonance, andcrystallographic electron microscopy. Preferably, the analog functionsas a dual function anti-glycPD-L1 antibody.

As used herein, the term “variant” when used in relation to a PD-L1polypeptide or to an anti-PD-L1 antibody refers to a polypeptide or ananti-PD-L1 antibody having one or more (such as, for example, about 1 toabout 25, about 1 to about 20, about 1 to about 15, about 1 to about 10,or about 1 to about 5) amino acid sequence substitutions, deletions,and/or additions as compared to a native or unmodified PD-L1 sequence oranti-PD-L1 antibody sequence. For example, a PD-L1 variant can resultfrom one or more (such as, for example, about 1 to about 25, about 1 toabout 20, about 1 to about 15, about 1 to about 10, or about 1 to about5 changes to an amino acid sequence of a native PD-L1. Also by way ofexample, a variant of an anti-PD-L1 antibody can result from one or more(such as, for example, about 1 to about 25, about 1 to about 20, about 1to about 15, about 1 to about 10, or about 1 to about 5 changes to anamino acid sequence of a native or previously unmodified anti-PD-L1antibody. Polypeptide variants can be prepared from the correspondingnucleic acid molecules encoding the variants. In certain embodiments,the variant is an anti-glycPD-L1 antibody that has one or more aminoacid substitutions, deletions or insertions in one or more CDR orframework regions and, preferably, the analog functions as a dualfunction anti-glycPD-L1 antibody.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) must be addressed by aparticular mathematical model or computer program (e.g., an“algorithm”). Methods that may be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, Lesk, A. M., Ed., 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,Smith, D. W., Ed., 1993, New York: Academic Press; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., 1994,New Jersey: Humana Press; Sequence Analysis in Molecular Biology, vonHeinje, G., 1987, New York: Academic Press; Sequence Analysis Primer,Gribskov, M. and Devereux, J., Eds., 1991, New York: M. Stockton Press;and Carillo et al., 1988, SIAM J. Applied Math., 48:1073.

In calculating percent identity, the sequences being compared can bealigned in a way that gives the largest match between the sequences. Anexample of a computer program that can be used to determine percentidentity is the GCG program package, which includes GAP (Devereux etal., 1984, Nucl. Acid Res., 12:387; Genetics Computer Group, Universityof Wisconsin, Madison, Wis.), which is a computer algorithm used toalign the two polypeptides or polynucleotides to determine their percentsequence identity. The sequences can be aligned for optimal matching oftheir respective amino acid or nucleotide sequences (the “matched span”as determined by the algorithm). A gap opening penalty (which iscalculated as 3 times the average diagonal, wherein the “averagediagonal” is the average of the diagonal of the comparison matrix beingused, and the “diagonal” is the score or number assigned to each perfectamino acid match by the particular comparison matrix; and a gapextension penalty (which is usually 1/10 times the gap opening penalty),as well as a comparison matrix such as PAM 250 or BLOSUM 62, are used inconjunction with the algorithm. In certain embodiments, a standardcomparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequenceand Structure, 5:345-352 for the PAM 250 comparison matrix; Henikoff etal., 1992, Proc. Natl. Acad. Sci. USA, 89:10915-10919 for the BLOSUM 62comparison matrix) is also used by the algorithm. Exemplary parametersfor determining percent identity for polypeptides or nucleotidesequences using the GAP program include the following: (i) Algorithm:Needleman et al., 1970, J. Mol. Biol., 48:443-453; (ii) Comparisonmatrix: BLOSUM 62 from Henikoff et al., Id.; (iii) Gap Penalty: 12 (butwith no penalty for end gaps); (iv) Gap Length Penalty: 4; and (v)Threshold of Similarity: 0.

Certain alignment schemes for aligning two amino acid sequences canresult in matching only a short region of the two sequences, and thissmall aligned region can have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (e.g., the GAPprogram) can be adjusted if so desired to result in an alignment thatspans a representative number of amino acids, for example, at least 50contiguous amino acids, of the target polypeptide.

Percent (%) amino acid sequence identity with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that is identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill of the practitioner in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared.

As used herein, the term “derivative” refers to a polypeptide thatcomprises an amino acid sequence of a reference polypeptide that hasbeen altered by the introduction of amino acid residue substitutions,deletions or additions. The reference polypeptide can be a PD-L1polypeptide or an anti-PD-L1 antibody. The term “derivative” as usedherein also refers to a PD-L1 polypeptide or an anti-PD-L1 antibody thathas been chemically modified, e.g., by the covalent attachment of anytype of molecule to the polypeptide. For example, a PD-L1 polypeptide oran anti-PD-L1 antibody can be chemically modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand, linkage to a peptide or proteintag molecule, or other protein, etc. The derivatives are modified in amanner that is different from the naturally occurring or startingpeptide or polypeptides, either in the type or location of the moleculesattached. Derivatives may further include deletion of one or morechemical groups which are naturally present on the peptide orpolypeptide. A derivative of a PD-L1 polypeptide or an anti-PD-L1antibody may be chemically modified by chemical modifications usingtechniques known to those of skill in the art, including, but notlimited to, specific chemical cleavage, acetylation, formulation,metabolic synthesis by tunicamycin, etc. Further, a derivative of aPD-L1 polypeptide or an anti-PD-L1 antibody can contain one or morenon-classical amino acids. A polypeptide derivative possesses a similaror identical function as the reference polypeptide, which can be a PD-L1polypeptide or an anti-PD-L1 antibody described herein, especially adual function anti-glycPD-L1 monoclonal antibody.

The term “fusion protein” as used herein refers to a polypeptide thatincludes amino acid sequences of at least two heterologous polypeptides.The term “fusion” when used in relation to a PD-L1 polypeptide or to ananti-PD-L1 antibody refers to the joining, fusing, or coupling of aPD-L1 polypeptide or an anti-PD-L1 antibody, variant and/or derivativethereof, with a heterologous peptide or polypeptide. In certainembodiments, the fusion protein retains the biological activity of thePD-L1 polypeptide or the anti-PD-L1 antibody. In certain embodiments,the fusion protein includes a PD-L1 antibody V_(H) region, V_(L) region,V_(H) CDR (one, two or three V_(H) CDRs), and/or V_(L) CDR (one, two orthree V_(L) CDRs) coupled, fused, or joined to a heterologous peptide orpolypeptide, wherein the fusion protein binds to an epitope on a PD-L1protein or peptide. In other embodiments, glycosylated PD-L1 peptidesfused to an Fc domain (preferably a human Fc domain) are provided.Fusion proteins may be prepared via chemical coupling reactions aspracticed in the art, or via molecular recombinant technology.

As used herein, the term “composition” refers to a product containingspecified component ingredients (e.g., a polypeptide or an antibodyprovided herein) in, optionally, specified or effective amounts, as wellas any desired product which results, directly or indirectly, from thecombination or interaction of the specific component ingredients in,optionally, the specified or effective amounts.

As used herein, the term “carrier” includes pharmaceutically acceptablecarriers, excipients, diluents, vehicles, or stabilizers that arenontoxic to the cell or mammal being exposed thereto at the dosages andconcentrations employed. Often, the physiologically acceptable carrieris an aqueous pH buffered solution. Examples of physiologicallyacceptable carriers include buffers such as phosphate, citrate,succinate, and other organic acids; antioxidants including ascorbicacid; low molecular weight (e.g., less than about 10 amino acidresidues) polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginineor lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, sucrose, or dextrins; chelating agents suchas EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,polyethylene glycol (PEG), and PLURONICS™. The term “carrier” can alsorefer to a diluent, adjuvant (e.g., Freund's adjuvant, complete orincomplete), excipient, or vehicle with which the therapeutic isadministered. Such carriers, including pharmaceutical carriers, can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is an exemplary carrier whena composition (e.g., a pharmaceutical composition) is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable excipients (e.g., pharmaceuticalexcipients) include, without limitation, starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. Compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Oral compositions,including formulations, can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in RemingtonPharmaceutical Sciences, 1990, Mack Publishing Co., Easton, Pa.Compositions, including pharmaceutical compounds, can contain atherapeutically effective amount of an anti-PD-L1 antibody, such as ananti-glycPD-L1 antibody, for example, in isolated or purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the subject (e.g., patient). The composition orformulation should suit the mode of administration.

As used herein, the term “excipient” refers to an inert substance whichis commonly used as a diluent, vehicle, preservative, binder, orstabilizing agent, and includes, but is not limited to, proteins (e.g.,serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid,lysine, arginine, glycine, histidine, etc.), fatty acids andphospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants(e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g.,sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol,sorbitol, etc.). See, also, for reference, Remington's PharmaceuticalSciences, Id., which is hereby incorporated by reference in itsentirety.

As used herein, the term “pharmaceutically acceptable” or“pharmacologically acceptable” refers to molecular entities,formulations and compositions that do not produce an adverse, allergic,or other untoward or unwanted reaction when administered, asappropriate, to an animal, such as a human. The preparation of apharmaceutical composition comprising an antibody or additional activeingredient are known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, Id. Moreover, for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by a regulatory agencyof the Federal or a state government, such as the FDA Office ofBiological Standards or as listed in the U.S. Pharmacopeia, EuropeanPharmacopeia, or other generally recognized Pharmacopeia for use inanimals, and more particularly, in humans.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient (e.g., an anti-PD-L1 antibody and an anti-glycPD-L1 antibody)to be effective, and which contains no additional components that wouldbe unacceptably toxic to a subject to whom the formulation would beadministered. Such a formulation can be sterile, i.e., aseptic or freefrom all living microorganisms and their spores, etc.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

As used herein, the term “treat,” “treatment,” or “treating” refers toadministration or application of a therapeutic agent to a subject inneed thereof, or performance of a procedure or modality on a subject,for the purpose of obtaining at least one positive therapeutic effect orbenefit, such as treating a disease or health-related condition. Forexample, a treatment can include administration of a pharmaceuticallyeffective amount of an antibody, or a composition or formulationthereof, that specifically binds to glycosylated PD-L1 for the purposeof treating various types of cancer. The terms “treatment regimen,”“dosing regimen,” or “dosing protocol,” are used interchangeably andrefer to the timing and dose of a therapeutic agent, such as ananti-glycPD-L1 antibody as described. As used herein, the term “subject”refers to either a human or a non-human animal, such as primates,mammals, and vertebrates having a cancer or diagnosed with a cancer. Inpreferred embodiments, the subject is a human. In some embodiments, thesubject is a cancer patient. In an embodiment, the subject in need willor is predicted to benefit from a dual function anti-glycPD-L1 antibodytreatment.

As used herein, the term “therapeutic benefit” or “therapeuticallyeffective” refers the promotion or enhancement of the well-being of asubject in need (e.g., a subject with a cancer or diagnosed with acancer) with respect to the medical treatment, therapy, dosageadministration, of a condition, particularly as a result of the use ofthe dual function anti-glycPD-L1 antibodies and the performance of thedescribed methods. This includes, but is not limited to, a reduction inthe frequency or severity of the signs or symptoms of a disease. Forexample, treatment of a cancer may involve, for example, a reduction inthe size of a tumor, a reduction in the invasiveness or severity of atumor, a reduction infiltration of cancer cells into a peripheral tissueor organ; a reduction in the growth rate of the tumor or cancer, or theprevention or reduction of metastasis. Treatment of cancer may alsorefer to achieving a sustained response in a subject or prolonging thesurvival of a subject with cancer.

As used herein, the term “administer” or “administration” refers to theact of physically delivering, e.g., via injection or an oral route, asubstance as it exists outside the body into a patient, such as by oral,subcutaneous, mucosal, intradermal, intravenous, intramuscular deliveryand/or any other method of physical delivery described herein or knownin the art. When a disease, disorder or condition, or a symptom thereof,is being treated therapeutically, administration of the substancetypically occurs after the onset of the disease, disorder or conditionor symptoms thereof. Prophylactic treatment involves the administrationof the substance at a time prior to the onset of the disease, disorderor condition or symptoms thereof.

As used herein, the term “effective amount” refers to the quantity oramount of a therapeutic (e.g., an antibody or pharmaceutical compositionprovided herein) which is sufficient to reduce, diminish, alleviate,and/or ameliorate the severity and/or duration of a cancer or a symptomrelated thereto. This term also encompasses an amount necessary for thereduction or amelioration of the advancement or progression of a cancer;the reduction or amelioration of the recurrence, development, or onsetof a cancer; and/or the improvement or enhancement of the prophylacticor therapeutic effect(s) of another cancer therapy (e.g., a therapyother than administration of an anti-PD-L1 antibody or anti-glycPD-L1antibody provided herein). In some embodiments, the effective amount ofan antibody provided herein is from about or equal to 0.1 mg/kg (mg ofantibody per kg weight of the subject) to about or equal to 100 mg/kg.In certain embodiments, an effective amount of an antibody providedtherein is about or equal to 0.1 mg/kg, about or equal to 0.5 mg/kg,about or equal to 1 mg/kg, about or equal to 3 mg/kg, about or equal to5 mg/kg, about or equal to 10 mg/kg, about or equal to 15 mg/kg, aboutor equal to 20 mg/kg, about or equal to 25 mg/kg, about or equal to 30mg/kg, about or equal to 35 mg/kg, about or equal to 40 mg/kg, about orequal to 45 mg/kg, about or equal to 50 mg/kg, about or equal to 60mg/kg, about or equal to 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.These amounts are meant to include amounts and ranges therein. In someembodiments, “effective amount” also refers to the amount of an antibodyprovided herein to achieve a specified result (e.g., preventing,blocking, or inhibiting cell surface PD-1 binding to cell surface PD-L1;or preventing, blocking, or inhibiting PD-1/PD-L1 mediatedimmunosuppression).

The term “in combination” in the context of the administration of othertherapies (e.g., other agents, cancer drugs, cancer therapies) includesthe use of more than one therapy (e.g., drug therapy and/or cancertherapy). Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (e.g., concurrent) andconsecutive administration in any order. The use of the term “incombination” does not restrict the order in which therapies areadministered to a subject. By way of nonlimiting example, a firsttherapy (e.g., agent, such as an anti-glycPD-L1 antibody) may beadministered before (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes,1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks,11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) theadministration of a second therapy (e.g., agent) to a subject having ordiagnosed with a cancer.

The combination of therapies (e.g., use of agents, including therapeuticagents) may be more effective than the additive effects of any two ormore single therapy (e.g., have a synergistic effect) or may have otherbenefits that are not predicted a priori such as improved side effectprofile, increased efficacy or duration of therapeutic effect, broaderpatient population in which the combination is effective, etc. Such aneffect is typically unexpected and cannot be predicted. For example, asynergistic effect of a combination of therapeutic agents frequentlypermits the use of lower dosages of one or more of the agents and/orless frequent administration of the agents to a cancer patient. Theability to utilize lower dosages of therapeutics and cancer therapiesand/or to administer the therapies less frequently reduces the potentialfor toxicity associated with the administration of the therapies to asubject without reducing the effectiveness of the therapies. Inaddition, a synergistic effect may result in improved efficacy oftherapies in the treatment or alleviation of a cancer. Also, asynergistic effect demonstrated by a combination of therapies (e.g.,therapeutic agents) may avoid or reduce adverse or unwanted side effectsassociated with the use of any single therapy.

Dual Function Anti-Glycosylated PD-L1 Antibodies

Provided herein are antibodies or binding fragments thereof that bind toglycosylated PD-L1 protein (e.g., a PD-L1 protein having a specificN-glycan structure; specific glycopeptides of PD-L1) or glycosylatedPD-L1 peptides and are dual function in that they reduce or block thebinding of PD-L1/PD-1 interaction and also promote internalization anddegradation of the PD-L1 on the tumor cell, as well as the use of suchantibodies in the treatment of disease, particularly cancer. Theantibodies preferentially bind to glycosylated PD-L1 as compared tounglycosylated PD-L1. The dual function anti-glycPD-L1 antibodies asdescribed herein may of the IgG, IgM, IgA, IgD, and IgE Ig classes, aswell as polypeptides comprising antibody CDR domains that retain antigenbinding activity. Illustratively, the dual function anti-glycPD-L1antibodies may be chimeric, affinity matured, humanized, or humanantibodies. In a preferred embodiment, the dual function anti-glycPD-L1antibodies are monoclonal antibodies. In certain embodiments, themonoclonal anti-glycPD-L1 antibodies are STM073 or STM108. In anotherpreferred embodiment, the dual function anti-glycPD-L1 antibody is ahumanized antibody or a chimeric antibody, particularly a humanized orchimeric form of STM073 or STM108. By known means and as describedherein, polyclonal or monoclonal antibodies, antibody fragments, bindingdomains and CDRs (including engineered forms of any of the foregoing)may be created that are specific for glycosylated PD-L1 antigen, one ormore of its respective epitopes, or conjugates of any of the foregoing,whether such antigens or epitopes are isolated from natural sources orare synthetic derivatives or variants of the natural compounds. Theantibodies may be bispecific or biparatopic.

In an embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences (e.g., V domains and/orCDRs) from a non-human donor grafted to a heterologous non-human, humanor humanized sequence (e.g., framework and/or constant domainsequences). In one embodiment, the non-human donor sequences are frommouse or rat. In specific embodiments the V_(H) and V_(L) domains arenon-human, e.g., murine and the constant domains are human. In oneembodiment, an antigen binding sequence is synthetic, e.g., obtained bymutagenesis (e.g., phage display screening of a human phage library,etc.). In one embodiment, a chimeric antibody has murine V regions andhuman C regions. In one embodiment, the murine light chain V region isfused to a human kappa light chain C region. In one embodiment, themurine heavy chain V region is fused to a human IgG1 C region.

In an embodiment, the antibody is an immunoglobulin single variabledomain derived from a camelid antibody, preferably from a heavy chaincamelid antibody, devoid of light chains, which are known as V_(H)Hdomain sequences or Nanobodies™. A Nanobody™ (Nb) is the smallestfunctional fragment or single variable domain (V_(H)H) of a naturallyoccurring single-chain antibody and is known to the person skilled inthe art. They are derived from heavy chain only antibodies seen incamelids (Hamers-Casterman et al., 1993, Nature, 363:446-448; Desmyteret al., 1996, Nat. Struct. Biol., p. 803-811). In the family of“camelids,” immunoglobulins devoid of light polypeptide chains arefound. “Camelids” comprise old world camelids (Camelus bactrianus andCamelus dromedarius) and new world camelids (for example, Lama paccos,Lama glama, Lama guanicoe and Lama vicugna). The single variable domainheavy chain antibody is herein designated as a Nanobody™ or a V_(H)Hantibody. The small size and unique biophysical properties of Nbs excelconventional antibody fragments for the recognition of uncommon orhidden epitopes and for binding into cavities or active sites of proteintargets. Further, Nbs can be designed as multi-specific and multivalentantibodies, attached to reporter molecules, or humanized. Nbs arestable, survive the gastro-intestinal system and can easily bemanufactured.

In another embodiment, the antibody is a bispecific antibody. Unifyingtwo antigen binding sites of different specificity into a singleconstruct, bispecific antibodies have the ability to bring together twodiscreet antigens with exquisite specificity and therefore have greatpotential as therapeutic agents. Bispecific antibodies were originallymade by fusing two hybridomas, each capable of producing a differentimmunoglobulin. Bispecific antibodies are also produced by joining twoscFv antibody fragments while omitting the Fc portion present in fullimmunoglobulins. Each scFv unit in such constructs can contain onevariable domain from each of the heavy (V_(H)) and light (V_(L))antibody chains, joined with one another via a synthetic polypeptidelinker, the latter often being genetically engineered so as to beminimally immunogenic while remaining maximally resistant toproteolysis. Respective scFv units may be joined by a number of knowntechniques, including incorporation of a short (usually less than 10amino acids) polypeptide spacer bridging the two scFv units, therebycreating a bispecific single chain antibody. The resulting bispecificsingle chain antibody is therefore a species containing two V_(H)/V_(L)pairs of different specificity on a single polypeptide chain, in whichthe V_(H) and V_(L) domains in a respective scFv unit are separated by apolypeptide linker long enough to allow intramolecular associationbetween these two domains, such that the so-formed scFv units arecontiguously tethered to one another through a polypeptide spacer keptshort enough to prevent unwanted association between, for example, theV_(H) domain of one scFv unit and the V_(L) of the other scFv unit.

In another embodiment, the antibody is a biparatopic antibody. As usedherein the term “biparatopic antibody” refers to a bispecific bindingmolecule that comprises two antigen binding domains which recognize andbind to two different non-overlapping epitopes, antigenic determinants,or domains on the same protein target, e.g., a tumor-associated PD-L1target antigen, or a glycosylated PD-L1 target antigen as describedherein. In an embodiment, a biparatopic antibody directed against aglycPD-L1 or one or more peptide portions thereof, as described herein,comprises a first immunoglobulin variable domain and a secondimmunoglobulin variable domain, wherein the two binding domains bind totwo different non-overlapping epitopes of the same target glycPD-L1protein. One or both of the epitopes recognized by the first and secondimmunoglobulin binding domains may be glycosylated or containglycosylated residues. Preferably, at least one of the immunoglobulinspreferentially binds the glycosylated form of the glycPD-L1 proteinrelative to the unglycosylated form.

In another embodiment, a biparatopic antibody comprises animmunoglobulin (preferably a tetravalent IgG) that binds to an epitopeon a glycPD-L1 target molecule and a scFv that binds to a different andnon-overlapping epitope on the same glycPD-L1 target molecule, in whichthe immunoglobulin and the scFv are linked by a linker so as to permitthe binding of the immunoglobulin and the scFv to the different andnon-overlapping epitopes on the glycPD-L1 target molecule. Accordingly,biparatopic antibodies are created from two anti-glycPD-L1 antibodiesthat bind to different epitopes (or domains) on the same glycPD-L1target (i.e., bi-paratopic binding) to enhance binding affinity/avidity;to increase antibody load on tumor cells for enhanced effectorfunctions, such as antibody dependent cellular cytotoxicity (ADCC)and/or complement dependent cytotoxicity (CDC); and/or to improve orincrease tumor retention time. In addition, bivalent biparatopicantibodies that target two non-overlapping epitopes on atumor-associated glycPD-L1 antigen have the potential to induceclustering of glycPD-L1 target molecules in the cell membrane, which, inturn, may promote increased internalization, lysosomal trafficking anddegradation. Biparatopic antibodies directed against two different,non-overlapping epitopes on a target protein/antigen may be generatedusing techniques known in the art. See, e.g., B. Roberts et al., 1999,Int. J. Cancer, Vol. 81:285-291 (carcinoembryonic antigen, CEA); D. Luet al., 1999, J. Immunol. Methods, Vol. 230:159-71 (vascular endothelialgrowth factor receptor 2, VEGF2); WO 2009/068627, Ablynx NV, publishedJun. 4, 2009; WO 2010/142534, and Ablynx NV, published Dec. 16, 2010.

In an embodiment, a bivalent biparatopic antibody may be produced byusing variable domain sequences from two different anti-glycPD-L1antibodies, identified as described herein, that recognize and bind todifferent non-overlapping epitopes on a given glycPD-L1 target protein,wherein the antibody contains the single-chain variable fragment (scFv)of one of the anti-glycPD-L1 antibodies attached to the N-terminus ofthe H chain and/or the L chain, or, alternatively, the C-terminus of theC_(H)3 domain, of the second anti-PD-L1 antibody that recognizes adifferent and non-overlapping epitope on glycPD-L1. The scFv may belinked to the second anti-PD-L1 antibody via a peptide linker, forexample, such as those used to link binding domains in an scFv. See,e.g., Dimasi et al., J. Mol. Biol., 393:672-692 (2009). The resultingbinding molecule product, or biparatopic antibody, contains fouranti-glycPD-L1 binding units, or two binding units on each arm of themolecule, that are able to interact with and bind to two differentepitopes on glycPD-L1. According to this embodiment, a bivalentbiparatopic antibody that targets two non-overlapping epitopes onglycPD-L1 expressed on the surface of a tumor cell could effectivelycrosslink the glycPD-L1 s through epitope binding to induce clusteringof glycPD-L1 on the cell surface, leading to the formation of largecomplexes that elicit and promote enhanced internalization and lysosomaldegradation. In an embodiment, the biparatopic antibody is linked to atoxin or anti-cancer drug to produce an antibody-drug conjugate (ADC) asdescribed further herein. The enhanced internalization and endocytosisof such anti-PD-L1 biparatopic antibodies and lysosomal traffickingultimately results in the delivery of greater amounts of toxin into thetarget cells and greater tumor cell killing or regression. Such effectswere observed both in vitro and in vivo as described for a biparatopicanti-HER2 ADC (J. Y. Li et al., 2016, Cancer Cell, Vol. 29:117-129).Illustratively, biparatopic anti-glycPD-L1 antibodies or anti-glycPD-L1ADCs may be produced that specifically bind to two non-overlappingepitopes of glycosylated membrane-bound PD-L1 to prevent or block itsinteraction with its PD-1 cognate binding partner and to promote theirinternalization and degradation, as well as killing of the tumor cellsif an anti-glycPD-L1 ADC is used. In particular embodiments, theanti-glycPD-L1 antibody is STM073, STM108, or ADC forms thereof.

In other embodiments, anti-glycPD-L1 binding molecules or antibodiesencompassed by the invention may be multiparatopic, i.e., containantigen binding domains which recognize and bind three, four, or moredifferent, preferably non-overlapping, epitopes or antigenicdeterminants on the same glycPD-L1 target molecule. In yet otherembodiments, the anti-glycPD-L1 antibodies are both bi- ormultiparatopic and multivalent, i.e., also comprise antigen bindingsites or “paratopes” that recognize and bind to one or more differentepitopes or antigenic determinants on different target glycPD-L1molecules.

Examples of antibody fragments suitable for use include, withoutlimitation: (i) the Fab fragment, consisting of V_(L), V_(H), C_(L), andC_(H1) domains; (ii) the “Fd” fragment consisting of the V_(H) andC_(H1) domains; (iii) the “Fv” fragment consisting of the V_(L) andV_(H) domains of a single antibody; (iv) the “dAb” fragment, whichconsists of a V_(H) domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments;(vii) single chain Fv molecules (“scFv”), in which a V_(H) domain and aV_(L) domain are linked by a peptide linker that allows the two domainsto associate to form a binding domain; (viii) bi-specific single chainFv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies,multivalent, or multispecific fragments constructed by gene fusion (U.S.Patent Appln. Pub. No. 20050214860). Fv, scFv, or diabody molecules maybe stabilized by the incorporation of disulfide bridges linking theV_(H) and V_(L) domains. Minibodies comprising a scFv joined to a C_(H3)domain (Hu et al., 1996, Cancer Res., 56:3055-3061) may also be useful.In addition, antibody-like binding peptidomimetics are also contemplatedin embodiments. “Antibody like binding peptidomimetics” (ABiPs), whichare peptides that act as pared-down antibodies and have certainadvantages of longer serum half-life as well as less cumbersomesynthesis methods, have been reported by Liu et al., 2003, Cell Mol.Biol., 49:209-216.

Animals may be inoculated with an antigen, such as a glycosylated PD-L1polypeptide or peptide to generate an immune response and produceantibodies specific for the glycosylated PD-L1 polypeptide. Frequently,an antigen is bound or conjugated to another molecule to enhance theimmune response. As used herein, a conjugate is any peptide,polypeptide, protein, or non-proteinaceous substance bound to an antigenthat is used to elicit an immune response in an animal. Antibodiesproduced in an animal in response to antigen inoculation comprise avariety of non-identical molecules (polyclonal antibodies) made from avariety of individual antibody producing B lymphocytes. A polyclonalantibody is a mixed population of antibody species, each of which mayrecognize a different epitope on the same antigen. Given the correctconditions for polyclonal antibody production in an animal, most of theantibodies in the animal's serum will recognize the collective epitopeson the antigenic compound to which the animal has been immunized. Thisspecificity is further enhanced by affinity purification to select onlythose antibodies that recognize the antigen or epitope of interest.

A monoclonal antibody is a single, clonal species of antibody whereinevery antibody molecule recognizes the same epitope because all antibodyproducing cells are derived from a single, antibody-producingB-lymphocyte. The methods for generating monoclonal antibodies (MAbs)generally begin along the same lines as those for preparing polyclonalantibodies. In some embodiments, rodents such as mice and rats are usedin generating monoclonal antibodies. In some embodiments, rabbit, sheep,or frog cells are used in generating monoclonal antibodies. The use ofrats is well known and may provide certain advantages. Mice (e.g.,BALB/c mice) are routinely used and generally give a high percentage ofstable fusions. Hybridoma technology as used in monoclonal antibodyproduction involves the fusion of a single, antibody-producing Blymphocyte isolated from a mouse previously immunized with aglycosylated PD-L1 protein or peptide with an immortalized myeloma cell,e.g., a mouse myeloma cell line. This technology provides a method topropagate a single antibody-producing cell for an indefinite number ofgenerations, such that unlimited quantities of structurally identicalantibodies having the same antigen or epitope specificity, i.e.,monoclonal antibodies, may be produced.

Engineered antibodies may be created using monoclonal and otherantibodies and recombinant DNA technology to produce other antibodies orchimeric molecules that retain the antigen or epitope bindingspecificity of the original antibody, i.e., the molecule has a specificbinding domain. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region or the CDRs of an antibody into thegenetic material for the framework regions, constant regions, orconstant regions plus framework regions, of a different antibody. See,for instance, U.S. Pat. Nos. 5,091,513 and 6,881,557, which areincorporated herein by reference.

By known means as described herein, polyclonal or monoclonal antibodies,antibody fragments having binding activity, binding domains and CDRs(including engineered forms of any of the foregoing), may be createdthat specifically bind to glycosylated PD-L1 protein, one or more of itsrespective epitopes, and have a dual function of blocking PD-L1 to PD-1binding and promoting internalization of PD-L1 in tumor cells, orconjugates of any of the foregoing, whether such antigens or epitopesare isolated from natural sources or are synthetic derivatives orvariants of the natural compounds.

Antibodies may be produced from any animal source, including birds andmammals. Preferably, the antibodies are ovine, murine (e.g., mouse andrat), rabbit, goat, guinea pig, camel, horse, or chicken. In addition,human antibodies can be obtained by screening human combinatorialantibody libraries. For example, bacteriophage antibody expressiontechnology allows specific antibodies to be produced in the absence ofanimal immunization, as described in U.S. Pat. No. 6,946,546, which isincorporated herein by reference. These techniques are further describedin Marks, 1992, Bio/Technol., 10:779-783; Stemmer, 1994, Nature,370:389-391; Gram et al., 1992, Proc. Natl. Acad. Sci. USA,89:3576-3580; Barbas et al., 1994, Proc. Natl. Acad. Sci. USA,91:3809-3813; and Schier et al., 1996, Gene, 169(2):147-155.

Methods for producing polyclonal antibodies in various animal species,as well as for producing monoclonal antibodies of various types,including humanized, chimeric, and fully human, are well known in theart and are highly reproducible. For example, the following U.S. patentsprovide descriptions of such methods and are herein incorporated byreference: U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797; 4,472,509;4,606,855; 4,703,003; 4,742,159; 4,767,720; 4,816,567; 4,867,973;4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066; 5,223,409;5,403,484; 5,420,253; 5,565,332; 5,571,698; 5,627,052; 5,656,434;5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861,155;5,871,907; 5,969,108; 6,054,297; 6,165,464; 6,365,157; 6,406,867;6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572;6,875,434; 6,891,024; 7,407,659; and 8,178,098.

It is expected that antibodies directed to glycosylated PD-L1 asprovided herein will have the ability to neutralize, block, inhibit, orcounteract the effects of glycosylated PD-L1 regardless of the animalspecies, monoclonal cell line or other source of the antibody. Certainanimal species may be less preferable for generating therapeuticantibodies because they may be more likely to cause an immune orallergic response due to activation of the complement system through the“Fc” portion of the antibody. However, whole antibodies may beenzymatically digested into the “Fc” (complement binding) fragment, andinto peptide fragments having the binding domains or CDRs. Removal ofthe Fc portion reduces the likelihood that this antibody fragment willelicit an undesirable immunological response and, thus, antibodieswithout an Fc portion may be preferential for prophylactic ortherapeutic treatments. As described above, antibodies may also beconstructed so as to be chimeric, humanized, or partially or fullyhuman, so as to reduce or eliminate potential adverse immunologicaleffects resulting from administering to an animal an antibody that hasbeen produced in, or has amino acid sequences from, another species.

Antibody proteins may be recombinant, or synthesized in vitro. It iscontemplated that in anti-glycPD-L1 antibody-containing compositions asdescribed herein there is between about 0.001 mg and about 10 mg oftotal antibody polypeptide per ml. Thus, the concentration of antibodyprotein in a composition can be about, at least about or at most aboutor equal to 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivabletherein). Of this, about, at least about, at most about, or equal to 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% may be an antibody that bindsglycosylated PD-L1.

An antibody or an immunological portion of an antibody that retainsbinding activity, can be chemically conjugated to, or recombinantlyexpressed as, a fusion protein with other proteins. For the purposes asdescribed herein, all such fused proteins are included in the definitionof antibodies or an immunological portion of an antibody. In someembodiments, the dual functional antibodies and antibody-like moleculesgenerated against glycosylated PD-L1, or polypeptides that are linked toat least one agent to form an antibody conjugate or payload areencompassed. In order to increase the efficacy of antibody molecules asdiagnostic or therapeutic agents, the antibody may be linked orcovalently bound or complexed with at least one desired molecule ormoiety to the antibody. Such a linked molecule or moiety may be, but isnot limited to, at least one effector or reporter molecule. Effectormolecules comprise molecules having a desired activity, e.g., cytotoxicactivity. Non-limiting examples of effector molecules that may beattached to antibodies include toxins, therapeutic enzymes, antibiotics,radio-labeled nucleotides and the like. By contrast, a reporter moleculeis defined as any moiety that may be detected using an assay.Non-limiting examples of reporter molecules that may be conjugated toantibodies include enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin, and the like. Several methods are known in theart for attaching or conjugating an antibody to a conjugate molecule ormoiety. Some attachment methods involve the use of a metal chelatecomplex, employing by way of nonlimiting example, an organic chelatingagent such a diethylenetriaminepentaacetic acid anhydride (DTPA);ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/ortetrachloro-3-6α-diphenylglycouril-3 attached to the antibody.Antibodies, particularly the antibodies as described herein, may also bereacted with an enzyme in the presence of a coupling agent such asglutaraldehyde or periodate. Conjugates with fluorescein markers areconventionally prepared in the presence of these coupling agents or byreaction with an isothiocyanate. In another embodiment, a dualfunctional anti-glycPD-L1 antibody as described herein may be coupled orlinked to a compound or substance, such as polyethylene glycol (PEG), toincrease its in vivo half-life in plasma, serum, or blood followingadministration.

Provided in a particular embodiment are antibodies, such as monoclonalantibodies, and humanized and chimeric forms thereof, and antigenbinding fragments thereof, that specifically and preferentially bindglycosylated PD-L1 protein relative to non-glycosylated PD-L1 proteinand have a dual activity in blocking PD-L1/PD-1 binding and promotingPD-L1 internalization and degradation. In an embodiment, the dualfunction anti-glycPD-L1 antibody specifically or preferentially binds toPD-L1 protein that is glycosylated at positions 35, 192, 200 and/or 219of the amino acid sequence of the PD-L1 protein, e.g., as set forth inSEQ ID NO: 1. For example, specific or selective binding of theanti-glycPD-L1 antibody involves binding of the antibody to glycosylatedPD-L1 antigen with a K_(d) less than half of the K_(d) exhibited relatedto unglycosylated PD-L1. In an embodiment, the anti-glycPD-L1 antibodybinds to glycosylated PD-L1 protein with a K_(d) at least 5 times lessthan the K_(d) exhibited relative to unglycosylated PD-L1. In anembodiment, the anti-glycPD-L1 antibody binds to glycosylated PD-L1protein with a K_(d) at least 10 times less than the K_(d) exhibitedrelative to unglycosylated PD-L1. In an embodiment, in a cell flowcytometry binding assay as described in Example 6, the antibody exhibitsbinding as expressed as MFI to cells expressing WT PD-L1 that is 1.5times, 2 times, 3, times, 4 times, 5 times, 6 times, 7 times, 8 times, 9times or 10 times greater than the MFI for binding to cells expressingunglycosylated PD-L1.

Provided in an embodiment is a dual function antibody or a bindingfragment thereof specific for and which preferentially binds toglycosylated PD-L1 that specifically binds the PD-L1 epitope bound bySTM073. In certain embodiments, the dual function anti-glycPD-L 1antibody binds an epitope on PD-L1 encompassing positions H69, Y112,R113 and K124 of the human PD-L1 amino acid sequence of SEQ ID NO: 1. Inan embodiment, the amino acids of the epitope are non-contiguous and theepitope is a conformational epitope. The regions of the human PD-L1polypeptide encompassing the STM073 MAb epitope have the sequenceVHGEEDLKVQH------DAGVYRCMISYGGADYKRITV (i.e., SEQ ID NO: 85 or V68-V128of SEQ ID NO: 1), in which the amino acid residues H69, Y112, R113 andK124, which comprise the epitope recognized by MAb STM073, areunderlined. In the STM073 epitope sequence, the dashes between aminoacid residue histidine (H) at position 78 and amino acid residueaspartic acid (D) at position 108 represent amino acids at positions79-107 of the human PD-L1 amino acid sequence of SEQ ID NO: 1.

Provided in another embodiment is a dual function antibody or a bindingfragment thereof specific for and that preferentially binds glycosylatedPD-L1 which is the anti-glycPD-L1 monoclonal antibody STM108, orhumanized or chimeric forms thereof. In specific embodiments, the dualfunction anti-glycPD-L1 specifically binds an epitope on PD-L1encompassing positions S80, Y81, K162 and S169 of the human PD-L1 aminoacid sequence of SEQ ID NO: 1 herein. The regions of the human PD-L1polypeptide encompassing the STM108 MAb epitope have the amino acidsequence LKVQHSSYRQR------EGYPKAEVIWTSSDHQ (i.e., L74-Q173 of SEQ ID NO:1), in which the amino acid residues S80, Y81, K162 and S169, comprisingthe epitope recognized by MAb STM108, are underlined. In the STM108epitope regions, the dashes between amino acid residue arginine (R) atposition 84 and amino acid residue glutamic acid (E) at position 158represent amino acids at positions 85-157 of the human PD-L1 amino acidsequence of SEQ ID NO: 1. Thus, the amino acids of the STM108 MAbepitope are non-contiguous, and the epitope is a conformational epitope.

The nucleic acid (DNA) and corresponding amino acid sequences of theheavy and light chain variable (V) domains of the STM073 MAb are shownin in Table 3 infra. Table 3 provides both the nucleotide and amino acidsequences of the mature (i.e., not containing the signal peptide) V_(H)and V_(L) domains of STM073 (SEQ ID NOS 2, 3, 10, and 11, respectively)and the V_(H) and V_(L) domain sequences containing the signal peptides(SEQ ID NOS: 87, 88, 89, and 90, respectively). In the heavy chain DNAand protein V domain sequences shown in Table 3 the amino terminalsignal sequence is represented in italicized font. Also shown in Table 3are the STM073 MAb heavy and light chain V domain CDRs, as determined byboth the Kabat and Chothia definitions.

In an embodiment, the dual function anti-glycPD-L1 antibody thatspecifically and preferentially binds glycosylated PD-L1 comprises aV_(H) domain of SEQ ID NO: 3 and a V_(L) domain of SEQ ID NO: 11. In anembodiment, the dual function anti-glycPD-L1 antibody competes forspecific binding to glycosylated PD-L1 with an antibody comprising aV_(H) domain of SEQ ID NO: 3 and a V_(L) domain of SEQ ID NO: 11. In anembodiment, the dual function anti-glycPD-L1 antibody that specificallyand preferentially binds glycosylated PD-L1 comprises a V_(H) domaincomprising Chothia CDRs 1-3 having amino acid sequences of SEQ ID NO: 4,SEQ ID NO: 6, and SEQ ID NO: 8, respectively, or Kabat CDRs 1-3 havingamino acid sequences of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9,respectively, or a combination thereof. In an embodiment, the dualfunction anti-glycPD-L1 antibody competes for specific binding toglycosylated PD-L1 with an antibody comprising a V_(H) domain comprisingChothia CDRs 1-3 having amino acid sequences of SEQ ID NO: 4, SEQ ID NO:6, and SEQ ID NO: 8, respectively, or Kabat CDRs 1-3 having amino acidsequences of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively,or a combination thereof. In an embodiment, the dual functionanti-glycPD-L1 antibody that specifically and preferentially bindsglycosylated PD-L1 comprises a V_(L) domain comprising CDRs 1-3 havingamino acid sequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16,respectively. In an embodiment, the dual function anti-glycPD-L1antibody competes for specific binding to glycosylated PD-L1 with anantibody comprising a V_(L) domain comprising CDRs 1-3 having amino acidsequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16,respectively. In an embodiment, the dual function anti-glycPD-L1antibody that specifically and preferentially binds glycosylated PD-L1comprises (a) a V_(H) domain comprising Chothia CDRs 1-3 having aminoacid sequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8,respectively, or Kabat CDRs 1-3 having amino acid sequences of SEQ IDNO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively, or a combinationthereof; and (b) a V_(L) domain comprising CDRs 1-3 having amino acidsequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16,respectively. In embodiments, the dual function anti-glycPD-L1 antibodycompetes for specific binding to glycosylated PD-L1 with an antibodycomprising the above-described V_(H) and V_(L) domains and the CDRstherein.

In an embodiment, the dual function anti-glycPD-L1 antibody thatspecifically binds glycosylated PD-L1 comprises a V_(H) domain that is80%, 85%, 90%, 95% 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 3 and/or a V_(L) domain that is 80%, 85%, 90%, 95% 98% or 99%identical to the amino acid sequence of SEQ ID NO: 11, and whichinhibits or blocks binding of glycosylated PD-L1 to PD-1 and promotesdestabilization of PD-L1 expression on the cell membrane. In anembodiment, the dual function anti-glycPD-L1 antibody that specificallyand preferentially binds glycosylated PD-L1 comprises a V_(H) domaincomprising CDRs 1-3 with at least 1, 2, or all 3 CDRs having at least 1,2, 3, 4 or 5 amino acid substitutions with respect to the amino acidsequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively,or amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9,respectively, which anti-glycPD-L1 antibody blocks binding ofglycosylated PD-L1 to PD-1 and promotes destabilization of expression ofPD-L1 on the cell membrane. In an embodiment, the dual functionanti-glycPD-L1 antibody that specifically and preferentially bindsglycosylated PD-L1 comprises a V_(L) domain comprising CDRs 1-3 with atleast 1, 2, or all 3 CDRs having at least 1, 2, 3, 4 or 5 amino acidsubstitutions with respect to amino acid sequences of SEQ ID NO: 12, SEQID NO: 14, and SEQ ID NO: 16, respectively. In an embodiment, the dualfunction anti-glycPD-L1 antibody that specifically and preferentiallybinds glycosylated PD-L1 comprises (a) a V_(H) domain comprising CDRs1-3 with at least 1, 2, or all 3 CDRs having at least 1, 2, 3, 4 or 5amino acid substitutions with respect to the amino acid sequences of SEQID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively, or amino acidsequences of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively;and (b) a V_(L) domain comprising CDRs 1-3 with at least 1, 2, or all 3CDRs having at least 1, 2, 3, 4 or 5 amino acid substitutions withrespect to amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 14, and SEQID NO: 16, respectively, which antibody blocks binding of glycosylatedPD-L1 to PD-1 and promotes destabilization of PD-L1 expression on thecell membrane. Also provided are humanized forms of STM073 using theAbM, Contact or IMGT defined CDRs, with human framework regions and,optionally, human constant domains.

The foregoing dual function anti-glycPD-L1 antibodies bind toglycosylated PD-L1 with a K_(d) less than half of the K_(d) exhibitedrelative to unglycosylated PD-L1. In an embodiment, the dual functionanti-glycPD-L1 antibodies bind to glycosylated PD-L1 protein with aK_(d) at least 5 times less than the K_(d) exhibited relative tounglycosylated PD-L1. In an embodiment, the dual function anti-glycPD-L1antibodies bind to glycosylated PD-L1 protein with a K_(d) at least 10times less than the K_(d) exhibited relative to unglycosylated PD-L1protein. In an embodiment, the binding affinity of the dual functionanti-glycPD-L1 antibody for glycosylated PD-L1 is from 5-20 nM or from5-10 nM inclusive of the lower and upper values. In an embodiment, in acell flow cytometry binding assay as described in Example 6, theantibody exhibits binding as expressed as MFI to cells expressing WTPD-L1 that is 1.5 times, 2 times, 3, times, 4 times, 5 times, 6 times, 7times, 8 times, 9 times or 10 times greater than the MFI for binding tocells expressing unglycosylated PD-L1.

In an embodiment, the antibody inhibits the interaction of PD-1 withPD-L1, and particularly inhibits the interaction of PD-1 expressed byeffector T-cells with PD-L1, particularly, glycosylated PD-L1, expressedby tumor cells. The antibody further promotes internalization of PD-L1and degradation of PD-L1, thereby reducing the level of PD-L1 on thecell surface.

In another particular embodiment, an antibody, or a binding fragmentthereof, is provided that specifically and preferentially bindsglycosylated PD-L1 which is the anti-glycPD-L1 monoclonal antibodySTM108. The nucleic acid (DNA) and corresponding amino acid sequences ofthe mature heavy and light chain variable (V) domains (SEQ ID NOs: 18,19, 26 and 27) of the STM108 MAb are shown in Table 3 infra. The DNA andamino acid sequences of the unprocessed heavy chain V domain (i.e.,containing a signal sequence at the N-terminal) are also shown in Table3 (SEQ ID NOs: 91 and 92, respectively). Also shown in Table 3 are theSTM108 MAb heavy and light chain V domain CDRs, according to both theKabat and Chothia definitions.

In an embodiment, the dual function anti-glycPD-L1 antibody thatspecifically and preferentially binds glycosylated PD-L1 comprises aV_(H) domain of the amino acid sequence of SEQ ID NO: 19 and a V_(L)domain of the amino acid sequence of SEQ ID NO: 27. In an embodiment,the dual function anti-glycPD-L1 antibody competes for specific bindingto glycosylated PD-L1 with an antibody comprising a V_(H) domain of theamino acid sequence of SEQ ID NO: 19 and a V_(L) domain of the aminoacid sequence of SEQ ID NO: 27. In an embodiment, the dual functionanti-glycPD-L1 antibody that specifically and preferentially bindsglycosylated PD-L1 comprises a V_(H) domain comprising Chothia CDRs1-3having amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 22, and SEQ IDNO: 24, respectively, or Kabat CDRs 1-3 having amino acid sequences ofSEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively, or acombination thereof. In an embodiment, the dual function anti-glycPD-L1antibody competes for specific binding to glycosylated PD-L1 with anantibody comprising a V_(H) domain comprising Chothia CDRs 1-3 withamino acid sequences of SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24,respectively, or Kabat CDRs 1-3 with amino acid sequences of SEQ ID NO:21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively, or a combinationthereof. In an embodiment, the dual function anti-glycPD-L1 antibodythat specifically and preferentially binds glycosylated PD-L1 comprisesa V_(L) domain comprising CDRs 1-3 having amino acid sequences of SEQ IDNO: 28, SEQ ID NO: 30, and SEQ ID NO: 32, respectively. In anembodiment, the dual function anti-glycPD-L1 antibody competes forspecific binding to glycosylated PD-L1 with an antibody comprising aV_(L) domain comprising CDRs 1-3 having amino acid sequences of SEQ IDNO: 28, SEQ ID NO: 30, and SEQ ID NO: 32, respectively. In anembodiment, the dual function anti-glycPD-L1 antibody that specificallyand preferentially binds glycosylated PD-L1 comprises (a) a V_(H) domaincomprising Chothia CDRs1-3 having amino acid sequences of SEQ ID NO: 20,SEQ ID NO: 22, and SEQ ID NO: 24, respectively, or Kabat CDRs 1-3 havingamino acid sequences of SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25,respectively, or a combination thereof; and (b) a V_(L) domaincomprising CDRs 1-3 having amino acid sequences of SEQ ID NO: 28, SEQ IDNO: 30, and SEQ ID NO: 32, respectively. In embodiments, theanti-glycPD-L1 antibody competes for specific binding to glycosylatedPD-L1 with an antibody comprising the above-described V_(H) and V_(L)domains and the CDRs therein.

In an embodiment, the dual function anti-glycPD-L1 antibody thatspecifically and preferentially binds glycosylated PD-L1 comprises aV_(H) domain that is 80%, 85%, 90%, 95% 98% or 99% identical to theamino acid sequence of SEQ ID NO: 19 and a V_(L) domain that is 80%,85%, 90%, 95% 98% or 99% identical to the amino acid sequence of SEQ IDNO: 27. In an embodiment, the dual function anti-glycPD-L1 antibody thatspecifically and preferentially binds glycosylated PD-L1 comprises aV_(H) domain comprising CDRs 1-3 with at least 1, 2, or all 3 CDRshaving at least 1, 2, 3, 4 or 5 amino acid substitutions with respect tothe amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO:24, respectively, or CDRs 1-3 having amino acid sequences of SEQ ID NO:21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively, or a combinationthereof. In an embodiment, the dual function anti-glycPD-L1 antibodythat specifically and preferentially binds glycosylated PD-L1 comprisesa V_(L) domain comprising CDRs 1-3 with at least 1, 2, or all 3 CDRshaving at least 1, 2, 3, 4 or 5 amino acid substitutions with respect tothe amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO:32, respectively. In an embodiment, the dual function anti-glycPD-L1antibody that specifically and preferentially binds glycosylated PD-L1comprises (a) a V_(H) domain comprising CDRs 1-3 with at least 1, 2, orall 3 CDRs having at least 1, 2, 3, 4 or 5 amino acid substitutions withrespect to the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 22, andSEQ ID NO: 24, respectively, or with respect to the amino acid sequencesof SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively, or acombination thereof; and/or (b) a V_(L) domain comprising CDRs 1-3 withat least 1, 2, or all 3 CDRs having at least 1, 2, 3, 4 or 5 amino acidsubstitutions with respect to the amino acid sequences of SEQ ID NO: 28,SEQ ID NO: 30, and SEQ ID NO: 32, respectively. Also provided arehumanized forms of STM108 using the AbM, Contact or IMGT defined CDRs,with human framework regions and, optionally, human constant domains.

In embodiments, the foregoing anti-glycPD-L1 antibodies bind toglycosylated PD-L1 with a K_(d) less than half of the K_(d) exhibitedrelative to unglycosylated PD-L1. In an embodiment, the anti-glycPD-L1antibodies bind to glycosylated PD-L1 protein with a K_(d) at least 5times less than the K_(d) exhibited relative to unglycosylated PD-L1. Inan embodiment, the anti-glycPD-L1 antibody binds to glycosylated PD-L1protein with a K_(d) at least 10 times less than the K_(d) exhibitedrelative to unglycosylated PD-L1 protein. In an embodiment, the bindingaffinity of STM108 MAb for glycosylated PD-L1 is from 5-20 nM or from5-10 nM inclusive of the lower and upper values. In an embodiment, in acell flow cytometry binding assay as described in Example 6, theantibody exhibits binding as expressed as MFI to cells expressing WTPD-L1 that is 1.5 times, 2 times, 3, times, 4 times, 5 times, 6 times, 7times, 8 times, 9 times or 10 times greater than the MFI for binding tocells expressing unglycosylated PD-L1. These anti-glycPD-L1 antibodiesinhibit the interaction of PD-1 with PD-L1, and particularly inhibit theinteraction of PD-1 expressed by effector T-cells with PD-L1,particularly, glycosylated PD-L1, expressed by tumor cells.

Also encompassed in an embodiment is an isolated dual functionanti-glycPD-L1 antibody that binds an epitope within the human PD-L1amino acid sequence DAGVYRCMISYGGADYKRITV (i.e., D108-V128 of SEQ ID NO:1). In an embodiment, an isolated dual function anti-glycPD-L1 antibodyspecifically binds a human PD-L1 epitope that is non-contiguous andencompasses positions Y112, R113 and S117 of the human PD-L1 amino acidsequence SEQ ID NO: 1 shown as the underlined amino acid residues in thefollowing sequence: DAGVYRCMISYGGADYKRITV (SEQ ID NO: 86).

Provided in an embodiment is an isolated dual function anti-glycPD-L1antibody comprising (a) a V_(H) domain comprising a Chothia CDR H1having an amino acid sequence of SEQ ID NO: 4 or a Kabat CDR H1 havingan amino acid sequence of SEQ ID NO: 5; a Chothia CDR H2 having an aminoacid sequence of SEQ ID NO: 6 or a Kabat CDR H2 having an amino acidsequence of SEQ ID NO: 7; and a Chothia CDR H3 having an amino acidsequence of SEQ ID NO: 8 or a Kabat CDR H3 having an amino acid sequenceof SEQ ID NO: 9; and a V_(L) domain comprising a CDR L1 having an aminoacid sequence of SEQ ID NO: 12; a CDR L2 having an amino acid sequenceof SEQ ID NO: 14; and a CDR L3 having an amino acid sequence of SEQ IDNO: 16. Also provided is an isolated dual function anti-glycPD-L1antibody that is a humanized form of STM073 having CDRs according to theIMGT, AbM or Contact definitions as detailed in Table 2, supra.

Provided in another embodiment is an isolated dual functionanti-glycPD-L1 antibody comprising (a) a V_(H) domain comprising aChothia CDR H1 having an amino acid sequence of SEQ ID NO: 20 or a KabatCDR H1 having an amino acid sequence of SEQ ID NO: 21; a Chothia CDR H2having an amino acid sequence of SEQ ID NO: 22 or a Kabat CDR H2 havingan amino acid sequence of SEQ ID NO: 23; and a Chothia CDR H3 having anamino acid sequence of SEQ ID NO: 24 or a Kabat CDR H3 having an aminoacid sequence of SEQ ID NO: 25; and a V_(L) domain comprising a CDR L1having an amino acid sequence of SEQ ID NO: 28; a CDR L2 having an aminoacid sequence of SEQ ID NO: 30; and a CDR L3 having an amino acidsequence of SEQ ID NO: 32. Also provided is an isolated dual functionanti-glycPD-L1 antibody that is a humanized form of STM108 having CDRsaccording to the IMGT, AbM or Contact definitions as detailed in Table2, supra.

Provided in another embodiment is an isolated dual functionanti-glycPD-L1 antibody, e.g., a monoclonal antibody, chimeric orhumanized form thereof, or binding fragment thereof, that specificallybinds to an epitope within an amino acid sequence selected fromVHGEEDLKVQH------DAGVYRCMISYGGADYKRITV (SEQ ID NO: 85),DAGVYRCMISYGGADYKRITV (SEQ ID NO: 86), orLKVQHSSYRQR------EGYPKAEVIWTSSDHQ (which are amino acids 74 to 84 and158 to 173, respectively, of SEQ ID NO: 1), which sequences are locatedwithin the human PD-L1 polypeptide sequence of SEQ ID NO: 1, i.e., themature PD-L1 protein comprising amino acids 19-290 of SEQ ID NO: 1. Inan embodiment, the antibody inhibits the interaction of PD-1 with PD-L1,and particularly inhibits the interaction of PD-1 expressed by effectorT-cells with PD-L1, particularly, glycosylated PD-L1, expressed by tumorcells, as well as facilitates internalization of PD-L1 from the cellmembrane into the cell and intracellular degradation of the PD-L1.

Provided in another embodiment is an isolated dual functionanti-glycPD-L1 antibody, e.g., a monoclonal antibody, chimeric orhumanized form thereof, or binding fragment thereof, that binds the sameepitope as MAb STM073, or an isolated anti-glycPD-L1 MAb as describedherein, wherein the epitope comprises amino acid residues H69, Y112,R113 and K124 of SEQ ID NO: 1. In another embodiment, an isolated dualfunction anti-glycPD-L1 antibody, e.g., a monoclonal antibody, chimericor humanized form thereof, or binding fragment thereof, that, when boundto glycosylated PD-L1, contacts at least one of the following amino acidresidues: H69, Y112, R113 and K124 of SEQ ID NO: 1 is provided. Inembodiments, the anti-glycPD-L1 antibody contacts at least two, at leastthree, or four of the amino acid residues comprising the epitoperegion(s) of PD-L1, i.e., glycosylated human PD-L1.

Provided in another embodiment is an isolated dual functionanti-glycPD-L1 antibody, e.g., a monoclonal antibody, chimeric orhumanized form thereof, or binding fragment thereof, that binds the sameepitope as MAb STM108, or an isolated anti-glycPD-L1 MAb which binds anepitope comprising amino acid residues S80, Y81, K162 and S169 of SEQ IDNO: 1. In another embodiment, an isolated dual function anti-glycPD-L1antibody that, when bound to glycosylated PD-L1, contacts at least oneof the following amino acid resides: S80, Y81, K162 and S169 of SEQ IDNO: 1 is provided.

Provided in another embodiment is an isolated dual functionanti-glycPD-L1 antibody, e.g., a monoclonal antibody, chimeric orhumanized form thereof, or binding fragment thereof, that, when bound toglycosylated PD-L1, contacts at least one of the following amino acidresidues: Y112, R113 and S117 of SEQ ID NO: 1.

Provided in another embodiment is an isolated dual functionanti-glycPD-L1 antibody, e.g., a monoclonal antibody, chimeric orhumanized form thereof, or binding fragment thereof, that, when bound toglycosylated PD-L1, contacts at least one amino acid within the aminoacid region from V68 to V128 of SEQ ID NO: 1, or within the amino acidregion from D108 to V128 of SEQ ID NO: 1, or within the amino acidregion from L74 to Q173 of SEQ ID NO: 1, or within the amino acidregions from L74 to R84 as well as from E158 to Q173 of SEQ ID NO: 1. Inanother embodiment, there is provided an isolated dual functionanti-glycPD-L1 antibody, e.g., a monoclonal antibody, chimeric orhumanized form thereof, or binding fragment thereof, that, when bound toglycosylated PD-L1, contacts at least one of the following group ofamino acid residues: H69, Y112, R113 and K124 within the amino acidregion from V68 to V128 of SEQ ID NO: 1. In another embodiment, there isprovided an isolated dual function anti-glycPD-L1 antibody, e.g., amonoclonal antibody, chimeric or humanized form thereof, or bindingfragment thereof, that, when bound to glycosylated PD-L1, contacts atleast one, at least two, at least three, or four of the following groupof amino acid residues: H69, S80, Y81, Y112, R113, K124, K162, S169within the amino acid region from V68 to V173 of SEQ ID NO: 1. In anembodiment, an isolated dual function anti-glycPD-L1 antibody, e.g., amonoclonal antibody, chimeric or humanized form thereof, or bindingfragment thereof, that, when bound to glycosylated PD-L1, contacts thefollowing group of amino acid residues: Y112, R113, S117 within theamino acid region from D108 to V128 of SEQ ID NO: 1 is provided.

Provided in another embodiment is an isolated dual functionanti-glycPD-L1 antibody, e.g., a monoclonal antibody, chimeric orhumanized form thereof, or binding fragment thereof, that, when bound toglycosylated PD-L1, binds at least amino acid region V68-V128 or atleast amino acid region D108-V128 of the PD-L1 protein (SEQ ID NO: 1),or a combination thereof.

Yet another embodiment provides an isolated nucleic acid comprising anucleotide sequence of SEQ ID NO: 2 or 18 encoding a V_(H) domain and anucleotide sequence of SEQ ID NO: 10 or 26 encoding an antibody V_(L)domain. In an embodiment, an isolated nucleotide sequence encoding ananti-glycPD-L1 antibody V_(H) domain, wherein the nucleotide sequence isat least 90-98% identical to the nucleotide sequence of SEQ ID NOs: 2 or18 is provided. In an embodiment, an isolated nucleotide sequenceencoding an anti-glycPD-L1 antibody V_(L) domain, wherein the nucleotidesequence is at least 90-98% identical to the nucleotide sequence of SEQID NOs: 19 or 26 is provided. In embodiments, the nucleotide sequencesencoding the V_(H) and/or the V_(L) domains are 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NOs: 2 or 18, orSEQ ID NOs: 10 or 26, respectively.

TABLE 3 Nucleotide and Amino Acid Sequences of anti-glycPD-L1 MAbsSEQ ID NO: Sequence Description SEQ ID NO: 2aagtgcagctggtggagtctgggggagccttagtg Nucleotide (DNA) sequenceaagcctggagggtccctgaaactctcctgtgcagc encoding mature STM073ctctggattcactttcagtaactctgccatgtctt heavy chain V domaingggttcgccagactccagagaagaggctggagtgg (Not including aminogtcgcaaccattagtagtgctggtagttataccta terminal nucleotides 1-58ctatccagacagtgtgaagggtcgattcaccatct encoding the signal sequence)ccagagacaatgccaagaacaccctgtacctgcaa atgagcagtctgaggtctgaggacacggccttgtattactgtacaagacattatgattactactttgact actggggccaaggcgccactctcacagtctcctcaSEQ ID NO: 3 EVQLVESGGALVKPGGSLKLSCAASGFTFSNSAMS Protein sequence of theWVRQTPEKRLEWVATISSAGSYTYYPDSVKGRFTI mature MAb STM073 heavySRDNAKNTLYLQMSSLRSEDTALYYCTRHYDYYFD chain V domain YWGQGATLTVSS(Not including amino terminal residues M1-C19which constitute the signal sequence) SEQ ID NO: 4 GFTFSNSMAb STM073 heavy chain V domain Chothia CDR1 SEQ ID NO: 5 NSAMSMAb STM073 heavy chain V domain Kabat CDR1 SEQ ID NO: 6 SSGGSYMAb STM073 heavy chain V domain Chothia CDR2 SEQ ID NO: 7TISSAGSYTYYPDSVKG MAb STM073 heavy chain V domain Kabat CDR2SEQ ID NO: 8 TRHYDYYFDY MAb STM073 heavy chain V domain Chothia CDR3SEQ ID NO: 9 TRHYDYYFDY MAb STM073 heavy chain V domain Kabat CDR3SEQ ID NO: 10 caaattgttctcacccagtctccagcaatcatgtcNucleotide (DNA) sequence tgcatctccaggggagaaggtcaccttgacctgcaencoding the mature STM073 gtgccagctcaagtgtaagttacatgcattggtackappa light chain V domain cagcagaagccaggatcctcccccagactcgtgat(Not including amino ttatgacacatccaacctggcttctggagtccctgterminal nucleotides 1-66 ttcgcttcagtggcagtgggtctgggacctcttacencoding the signal sequence) tctctcacagtcagccgaatggaggctgaagatgctgccacttattactgccagcagtggagtgatcacc cgctcacgttcggtgctgggaccaagctggagctgaaac SEQ ID NO: 11 QIVLTQSPAIMSASPGEKVTLTCSASSSVSYMHWYProtein sequence of the QQKPGSSPRLVIYDTSNLASGVPVRFSGSGSGTSYmature MAb STM073 kappa SLTVSRMEAEDAATYYCQQWSDHPLTFGAGTKLELlight chain V domain K (Not including amino terminal residues M1-G22which constitute the signal sequence) SEQ ID NO: 12 SASSSVSYMHMAb STM073 kappa light chain V domain Chothia CDR1 SEQ ID NO: 13SASSSVSYMH MAb STM073 kappa light chain V domain Kabat CDR1SEQ ID NO: 14 DTSNLAS MAb STM073 kappa light chain V domain Chothia CDR2SEQ ID NO: 15 DTSNLAS MAb STM073 kappa light chain V domain Kabat CDR2(f SEQ ID NO: 16 QQWSDHPLT MAb STM073 kappa light chain V domain ChothiaCDR3 SEQ ID NO: 17 QQWSDHPLT MAb STM073 kappa lightchain V domain Kabat CDR3 SEQ ID NO: 18gaagtgatgctggtggagtctgggggagccttagtgaa Nucleotide (DNA) sequencegcctggagggtccctgaaactctcctgtgcagcttctg encoding the mature MAbgattcagtttgagtaactatgtcatgtcttgggttcgc STM108 heavy chain Vcagactccagagaagaggctggagtgggtcgcaaccat domaintagtagtggtggtaggtatatctactatacagacagtg (Not including aminotgaagggtcgattcaccatctccagggacaatgccagg terminal nucleotides 1-57aacaccctgtacctgcaaatgagcagtctgaggtctga encoding the signal sequence)ggacacggccatgtattattgtgcaagagacggtagtaccttgtactactttgactattggggccaaggcaccact ctcacagtctcctca SEQ ID NO: 19EVMLVESGGALVKPGGSLKLSCAASGFSLSNYVMS Protein sequence of theWVRQTPEKRLEWVATISSGGRYIYYTDSVKGRFTI mature MAb STM108 heavySRDNARNTLYLQMSSLRSEDTAMYYCARDGSTLYY chain V domain FDYWGQGTTLTVSS(Not including amino terminal residues M1-C19which constitute the signal sequence) SEQ ID NO: 20 GFSLSNYMAb STM108 heavy chain V domain Chothia CDR1 SEQ ID NO: 21 NYVMSMAb STM108 heavy chain V domain Kabat CDR1 SEQ ID NO: 22 SSGGRYMAb STM108 heavy chain V domain Chothia CDR2 SEQ ID NO: 23TISSGGRYIYYTDSVKG MAb STM108 heavy chain V domain Kabat CDR2SEQ ID NO: 24 DGSTLYYFDY MAb STM108 heavy chain V domain Chothia CDR3SEQ ID NO: 25 DGSTLYYFDY MAb STM108 heavy chain V domain Kabat CDR3SEQ ID NO: 26 caagtgcagattttcagcttcctgctaatcagtgcctcNucleotide sequence (DNA) agtcatactgtccagaggacaaactgttctcacccagtencoding the mature MAb ctccagcaatcatgtctgcatctccaggggagaaggtcSTM108 kappa light chain V accatgacctgcagtgccagctcaagtgtagattacat domaingtactggtaccagcagaagccaggatcctcccccagactcctgatttatgacacatccaacctggcttctggagtccctgttcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattactgccagcagtggagtagttccccacccatcacgttcggtactgggaccaaggtggagctgaaa SEQ ID NO: 27QVQIFSFLLISASVILSRGQTVLTQSPAIMSASPG Protein sequence of theEKVTMTCSASSSVDYMYWYQQKPGSSPRLLIYDTS mature MAb STM108 kappaNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYY light chain V domainCQQWSSSPPITFGTGTKVELK SEQ ID NO: 28 SASSSVDYMY MAb STM108 kappa lightchain V domain Chothia CDR1 SEQ ID NO: 29 SASSSVDYMYMAb STM108 kappa light chain V domain Kabat CDR1 SEQ ID NO: 30 DTSNLASMAb STM108 kappa light chain V domain Chothia CDR2 SEQ ID NO: 31 DTSNLASMAb STM108 kappa light chain V domain Kabat CDR2 SEQ ID NO: 32QQWSSSPPIT MAb STM108 kappa light chain V domain Chothia CDR3SEQ ID NO: 33 QQWSSSPPIT MAb STM108 kappa lightchain V domain Kabat CDR3 SEQ ID NO: 87

Nucleotide (DNA) sequence

aagtgcagctgg encoding the STM073 heavytggagtctgggggagccttagtgaagcctggaggg chain V domain, includingtccctgaaactctcctgtgcagcctctggattcac amino terminal nucleotides 1-tttcagtaactctgccatgtcttgggttcgccaga 58 (in italics) which encodectccagagaagaggctggagtgggtcgcaaccatt the signal sequenceagtagtgctggtagttatacctactatccagacag tgtgaagggtcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctg aggtctgaggacacggccttgtattactgtacaagacattatgattactactttgactactggggccaag gcgccactctcacagtctcctcaSEQ ID NO: 88

CEVQLVESGGALVKPGG Protein sequence of the MAbSLKLSCAASGFTFSNSAMSWVRQTPEKRLEWVATI STM073 heavy chain VSSAGSYTYYPDSVKGRFTISRDNAKNTLyLQMSSL domain, including aminoRSEDTALYYCTRHYDYYFDYWGQGATLTVSS terminal residues M1-C19 (initalics) which constitute the signal sequence SEQ ID NO: 89

Nucleotide (DNA) sequence

caaa encoding the STM073 kappa ttgttctcacccagtctccagcaatcatgtctgcalight chain V domain, tctccaggggagaaggtcaccttgacctgcagtgcincluding amino terminal cagctcaagtgtaagttacatgcattggtaccagcnucleotides 1-66 (in italics) agaagccaggatcctcccccagactcgtgatttatwhich encode the signal gacacatccaacctggcttctggagtccctgttcg sequencecttcagtggcagtgggtctgggacctcttactctc tcacagtcagccgaatggaggctgaagatgctgccacttattactgccagcagtggagtgatcacccgct cacgttcggtgctgggaccaagctggagctgaaacSEQ ID NO: 90

QIVLTQSPAIMSA Protein sequence of the MAbSPGEKVTLTCSASSSVSYMHWYQQKPGSSPRLVIY STM073 kappa light chain VDTSNLASGVPVRFSGSGSGTSYSLTVSRMEAEDAA domain, including aminoTYYCQQWSDHPLTFGAGTKLELK terminal residues M1-G22 (initalics) which constitute the signal sequence SEQ ID NO: 91

Nucleotide (DNA) sequence

gaagtgatgctggtggagt encoding the STM108 heavyctgggggagccttagtgaagcctggagggtccctgaaa chain V domain, includingctctcctgtgcagcttctggattcagtttgagtaacta amino terminal nucleotides 1-tgtcatgtcttgggttcgccagactccagagaagaggc 57 (in italics) which encodetggagtgggtcgcaaccattagtagtggtggtaggtat the signal sequence of theatctactatacagacagtgtgaagggtcgattcaccat STM108 proteinctccagggacaatgccaggaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattattgtgcaagagacggtagtaccttgtactactttgactattggggccaaggcaccactctcacagtctcctca SEQ ID NO: 92

EVMLVESGGALVKPGG Protein sequence of the MAbSLKLSCAASGFSLSNYVMSWVRQTPEKRLEWVATI STM108 heavy chain VSSGGRYIYYTDSVKGRFTISRDNARNTLYLQMSSL domain, including aminoRSEDTAMYYCARDGSTLYYFDYWGQGTTLIVSS terminal residues M1-C19 (initalics) which constitute the signal sequence

Disease Treatment

In certain aspects, an antibody or antigen binding fragment thereof, asdescribed in the embodiments herein (e.g., a dual function antibody thatspecifically binds to glycosylated PD-L1) may be administered to treat acancer in a subject suffering therefrom or to prevent cancer in asubject with a predisposition (such as a genetic predisposition, priorenvironmental exposure to a carcinogen, prior incidence of cancer, etc.)to develop a cancer. Accordingly, provided herein are methods oftreating a cancer by administering to a subject in need atherapeutically effective amount of at least one dual functionanti-glycPD-L1 antibody to treat the cancer. As noted herein, treatmentinvolves reducing, preventing, inhibiting, or blocking the growth,proliferation, migration, etc. of cancer cells, and includes causingcell killing or apoptosis of cancer cells. The treatment may alsoprevent or cause regression of metastasis of tumor cells. The methodsdescribed herein provide a benefit to the subject, e.g., a humanpatient, undergoing treatment, with particular regard to tumor cellsthat express glycosylated PD-L1 cell surface proteins that canbind/interact with PD-1 expressed on the cell surface of immune effectorcells, such as T-cells, particularly, killer or cytotoxic T-cells.

Treatment of these subjects with an effective amount of at least one ofthe dual function anti-glycPD-L1 antibodies as described is expected toresult in binding of the antibody(ies) to glycosylated PD-L1 on thetumor cells and preventing, blocking, or inhibiting the interaction ofPD-L1 and PD-1 and promoting the internalization and degradation ofPD-L1 to prevent, block or inhibit the interaction of thePD-L1-expressing tumor cells with PD-1-expressing T cells, therebypreventing or avoiding immunosuppression of T-cell activity and allowingT cells to be activated to kill the PD-L1-bearing tumor cells. In apreferred embodiment, the dual function anti-PD-L1 antibody binds tohuman PD-L1 and the subject is a human patient. Accordingly, the methodsprovided herein are advantageous for a subject who is in need of,capable of benefiting from, or who is desirous of receiving the benefitof, the anti-cancer results achieved by the practice of the presentmethods. A subject's seeking the therapeutic benefits of the methodsinvolving administration of at least one dual function anti-glycPD-L1antibody in a therapeutically effective amount, or receiving suchtherapeutic benefits offer advantages to the art. In addition, thepresent methods offer the further advantages of eliminating or avoidingside effects, adverse outcomes, contraindications, and the like, orreducing the risk or potential for such issues to occur compared withother treatments and treatment modalities.

In specific embodiments, the subject suffering from or predisposed tocancer is administered a therapeutically effective amount of a humanizedor chimeric form of STM073 or STM108 that is a dual functionanti-glycPD-L1 antibody. It may be advantageous to administer more thanone type of anti-glycPD-L1 antibody for the treatment of cancer. Forexample, chimeric or humanized forms of both STM073 and STM108 may beco-administered to a patient in need thereof. Alternatively, a chimericor humanized form of STM073 may be co-administered with anotheranti-glycPD-L1 antibody to a patient in need thereof or a chimeric orhumanized form of STM108 may be co-administered with anotheranti-glycPD-L1 antibody to a patient in need thereof. Co-administrationof the anti-glycPD-L1 antibodies may be more therapeutically orprophylactically effective than administration of either antibody aloneand/or may permit administration of a lower dose or with lower frequencythan either antibody alone.

Cancers for which the present treatment methods are useful include anymalignant cell type, such as those found in a solid tumor or ahematological tumor, particularly tumors with cells that expressglycosylated PD-L1 on their surface. In general, a tumor refers to amalignant or a potentially malignant neoplasm or tissue mass of anysize, and includes primary tumors and secondary tumors. A solid tumor isan abnormal tissue mass or growth that usually does not contain cysts orliquid. Exemplary solid tumors can include, but are not limited to, atumor of an organ selected from the group consisting of pancreas, gallbladder, colon, cecum, stomach, brain, head, neck, ovary, testes,kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary hematological tumors include tumors of the bone marrow, T or Bcell malignancies, leukemias, lymphomas, blastomas, myelomas, and thelike. Further examples of cancers that may be treated using the methodsprovided herein include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer(including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer and gastrointestinal stromal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, varioustypes of head and neck cancer, melanoma, superficial spreading melanoma,lentigo malignant melanoma, acral lentiginous melanomas, nodularmelanomas, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairycell leukemia, multiple myeloma, acute myeloid leukemia (AML) andchronic myeloblastic leukemia.

The cancer may specifically be of the following histological types,though it need not be limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; malignantmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; paragranuloma; malignant lymphoma, smalllymphocytic; malignant lymphoma, large cell, diffuse; malignantlymphoma, follicular; mycosis fungoides; other specified non-Hodgkin'slymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma;immunoproliferative small intestinal disease; leukemia; lymphoidleukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cellleukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia;monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia;myeloid sarcoma; and hairy cell leukemia.

The cancer to be treated preferably is positive for PD-L1, particularlyglycosylated PD-L1. In certain embodiments, the tumor cells are alsopositive for a tumor cell marker such as EGFR or HER2/neu expression,e.g., as expressed on breast cancer cells. The presence or absence ofthese markers may indicate that combination therapy with a targetedtherapeutic, such as a tyrosine kinase inhibitor, e.g., gefitinib for anEGFR-positive cancer, or Herceptin for a HER2/neu-positive cancer, incombination with the dual function anti-glycPD-L1 antibodies asdescribed herein, would provide a treatment benefit for a subject inneed. In certain embodiments, the cancer is a BLBC.

Other markers that may be used to characterize cancers to guide choiceof therapy or monitor therapy include ALK gene rearrangements andoverexpression in non-small cell lung cancer and anaplastic large celllymphoma; alpha-fetoprotein (AFP) for liver cancer and germ cell tumors;beta-2-microglobulin (B2M) for multiple myeloma, chronic lymphocyticleukemia, and some lymphomas; beta-human chorionic gonadotropin(Beta-hCG) for choriocarcinoma and germ cell tumors; BRCA1 and BRCA2gene mutations for ovarian cancer and breast cancer; BCR-ABL fusion gene(Philadelphia chromosome) for chronic myeloid leukemia, acutelymphoblastic leukemia, and acute myelogenous leukemia; BRAF V600mutations for cutaneous melanoma and colorectal cancer; C-kit/CD117 forgastrointestinal stromal tumor and mucosal melanoma; CA15-3/CA27.29 forbreast cancer; CA19-9 for pancreatic cancer, gallbladder cancer, bileduct cancer, and gastric cancer; CA-125 for ovarian cancer; calcitoninfor medullary thyroid cancer; carcinoembryonic antigen (CEA) forcolorectal cancer and some other cancers; CD20 for non-Hodgkin lymphoma;Chromogranin A (CgA) for neuroendocrine tumors; chromosomes 3, 7, 17,and 9p21 for bladder cancer; cytokeratin fragment 21-1 for lung cancer;EGFR gene mutation analysis for non-small cell lung cancer; estrogenreceptor (ER)/progesterone receptor (PR) for breast cancer;fibrin/fibrinogen for bladder cancer; HE4 for ovarian cancer; HER2/neugene amplification or protein overexpression for breast cancer, gastriccancer, and gastroesophageal junction adenocarcinoma; immunoglobulinsfor multiple myeloma and Waldenström macroglobulinemia; KRAS genemutation analysis for colorectal cancer and non-small cell lung cancer;lactate dehydrogenase for germ cell tumors, lymphoma, leukemia,melanoma, and neuroblastoma; neuron-specific enolase (NSE) for smallcell lung cancer and neuroblastoma; nuclear matrix protein 22 forbladder cancer; prostate-specific antigen (PSA) for prostate cancer;thyroglobulin for thyroid cancer; and urokinase plasminogen activator(uPA) and plasminogen activator inhibitor (PAI-1) for breast cancer.

The dual function anti-glycPD-L1 antibodies may be used as antitumoragents in a variety of modalities. A particular embodiment relates tomethods of using an antibody as an antitumor agent, and thereforecomprises contacting a population of tumor cells with a therapeuticallyeffective amount of the antibody, or a composition containing theantibody, for a time period sufficient to block or inhibit tumor cellgrowth or to effect apoptosis of the tumor cells. In an embodiment,contacting a tumor cell in vivo is accomplished by administering to apatient in need, for example, by intravenous, subcutaneous,intraperitoneal, or intratumoral injection, a therapeutically effectiveamount of a physiologically tolerable composition comprising a dualfunction anti-glycPD-L1 antibody as described herein. The antibody maybe administered parenterally by injection or by gradual infusion overtime. Useful administration and delivery regimens include intravenous,intraperitoneal, oral, intramuscular, subcutaneous, intracavity,intrathecal, transdermal, dermal, peristaltic means, or direct injectioninto the tissue containing the tumor cells.

Therapeutic compositions comprising antibodies are conventionallyadministered intravenously, such as by injection of a unit dose, forexample. The term “unit dose” when used in reference to a therapeuticcomposition refers to physically discrete units suitable as unitarydosage for the subject, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required diluent, i.e., carrier, or vehicle. Thedual function anti-glycPD-L1 antibody containing compositions areadministered in a manner compatible with the dosage formulation, and ina therapeutically effective amount. The quantity to be administereddepends on the subject to be treated, capacity of the subject's systemto utilize the active ingredient, and degree of therapeutic effectdesired. Precise amounts of active ingredient to be administered dependon the judgment of the practitioner and are peculiar to each individual.However, suitable dosage ranges for systemic application are disclosedherein and depend on the route of administration. Suitable regimens forinitial and booster administration are also contemplated and maytypically involve an initial administration followed by repeated dosesat one or more intervals (hours) by a subsequent injection or otheradministration. Exemplary multiple administrations are suitable formaintaining continuously high serum and tissue levels of antibody.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated.

It is contemplated that an anti-glycPD-L1 antibody may be administeredsystemically or locally to treat disease, such as to inhibit tumor cellgrowth or to kill cancer cells in cancer patients with locally advancedor metastatic cancers. The antibodies may be administered alone or incombination with anti-proliferative drugs or anticancer drugs. In anembodiment, the anti-glycPD-L1 antibodies are administered to reduce thecancer load in the patient prior to surgery or other procedures.Alternatively, they can be administered at periodic intervals aftersurgery to ensure that any remaining cancer (e.g., cancer that thesurgery failed to eliminate) is reduced in size or growth capacityand/or does not survive. As noted hereinabove, a therapeuticallyeffective amount of an antibody is a predetermined amount calculated toachieve the desired effect. Thus, the dosage ranges for theadministration of an anti-glycPD-L1 antibody are those large enough toproduce the desired effect in which the symptoms of tumor cell divisionand cell cycling are reduced. Optimally, the dosage should not be solarge as to cause adverse side effects, such as hyperviscositysyndromes, pulmonary edema, congestive heart failure, neurologicaleffects, and the like. Generally, the dosage will vary with age of,condition of, size and gender of, and extent of the disease in thepatient and can be determined by one of skill in the art such as amedical practitioner or clinician. Of course, the dosage may be adjustedby the individual physician in the event of any complication.

Treatment Methods

In certain embodiments, the compositions and methods as describedinvolve the administration of a dual function anti-glycPD-L1 antibody,alone, or in combination with a second or additional drug or therapy.Such drug or therapy may be applied in the treatment of any disease thatis associated with PD-L1 or glycosylated PD-L1, preferably with theinteraction of human PD-L1 or glycosylated human PD-L1 with human PD-1.For example, the disease may be a cancer. The compositions and methodscomprising at least one anti-PD-L1 antibody that preferentially binds toglycosylated PD-L1 protein and both blocks or inhibits PD-L1 to PD-1binding and promotes PD-L1 internalization and degradation, or a bindingportion thereof, have a therapeutic or protective effect in thetreatment of a cancer or other disease, particularly by preventing,reducing, blocking, or inhibiting the PD-1/PD-L1 interaction, therebyproviding a therapeutic effect and treatment.

The compositions and methods, including combination therapies, have atherapeutic or protective effect and may enhance the therapeutic orprotective effect, and/or increase the therapeutic effect of anotheranti-cancer or anti-hyperproliferative therapy. Therapeutic andprophylactic methods and compositions can be provided in a combinedamount effective to achieve the desired effect, such as the killing of acancer cell and/or the inhibition of cellular hyperproliferation. Thisprocess may involve administering a dual function anti-glycPD-L 1antibody or a binding fragment thereof and a second therapy. The secondtherapy may or may not have a direct cytotoxic effect. For example, thesecond therapy may be an agent that upregulates the immune systemwithout having a direct cytotoxic effect. A tissue, tumor, and/or cellcan be exposed to one or more compositions or pharmacologicalformulation(s) comprising one or more of the agents (e.g., an antibodyor an anti-cancer agent), or by exposing the tissue, tumor, and/or cellwith two or more distinct compositions or formulations, wherein onecomposition provides, for example, 1) an antibody, 2) an anti-canceragent, 3) both an antibody and an anti-cancer agent, or 4) two or moreantibodies. In some embodiments, the second therapy is also ananti-PD-L1 antibody, preferably an anti-glycPD-L1 antibody thatpreferentially binds glycosylated PD-L1 versus unglycosylated PD-L1 or,in other embodiments, an anti-PD-1 antibody. Without limitation,exemplary anti-PD-1 antibodies include pembrolizumab and nivolumab;exemplary anti-PD-L1 antibodies include atezolizumab. Also, it iscontemplated that such a combination therapy can be used in conjunctionwith chemotherapy, radiotherapy, surgical therapy, or immunotherapy.

By way of example, the terms “contacted” and “exposed,” when applied toa cell, are used herein to describe a process by which a therapeuticpolypeptide, preferably an anti-glycPD-L1 antibody as described herein,is delivered to a target cell or is placed in direct juxtaposition withthe target cell, particularly to bind specifically to the targetantigen, e.g., PD-L1, particularly, glycosylated PD-L1, expressed orhighly expressed on the surface of tumor or cancer cells. Such bindingby a therapeutic anti-glycPD-L1 antibody or binding fragment thereofprevents, blocks, inhibits, or reduces the interaction of the tumor orcancer cell-expressed PD-L1 with PD-1 on an effector T-cell, therebypreventing immunosuppression associated with the PD-LI/PD-1 interaction.In embodiments, a chemotherapeutic or radiotherapeutic agent are alsoadministered or delivered to the subject in conjunction with theanti-glycPD-L1 antibody or binding fragment thereof. To achieve cellkilling, for example, one or more agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

An anti-glycPD-L1 antibody may be administered before, during, after, orin various combinations relative to another anti-cancer treatment. Theadministrations may be in intervals ranging from concurrently to minutesto days to weeks before or after one another. In embodiments in whichthe antibody is provided to a patient separately from an anti-canceragent, it would be generally ensured that a significant period of timedid not expire between the time of each delivery, such that theadministered compounds would still be able to exert an advantageouslycombined effect for the patient. Illustratively, in such instances, itis contemplated that one may provide a patient with the antibody and theanti-cancer therapy within about 12 to 24 or 72 h of each other and,more particularly, within about 6-12 h of each other. In some situationsit may be desirable to extend the time period for treatmentsignificantly where several days (2, 3, 4, 5, 6, or 7) to several weeks(1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

In certain embodiments, a course of treatment or treatment cycle willlast 1-90 days or more (this range includes intervening days and thelast day). It is contemplated that one agent may be given on any day ofday 1 to day 90 (this such range includes intervening days and the lastday) or any combination thereof, and another agent is given on any dayof day 1 to day 90 (this such range includes intervening days and thelast day) or any combination thereof. Within a single day (24-hourperiod), the patient may be given one or multiple administrations of theagent(s). Moreover, after a course of treatment, it is contemplated thatthere may be a period of time at which no anti-cancer treatment isadministered. This time period may last, for example, for 1-7 days,and/or 1-5 weeks, and/or 1-12 months or more (this such range includesintervening days and the upper time point), depending on the conditionof the patient, such as prognosis, strength, health, etc. Treatmentcycles would be repeated as necessary. Various combinations oftreatments may be employed. In the representative examples ofcombination treatment regimens shown below, an antibody, such as ananti-glycPD-L1 antibody or binding fragment thereof is represented by“A” and an anti-cancer therapy is represented by “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A.

Administration of any antibody or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring adverseevents and toxicity, particularly those that may be attributable tocombination therapy.

In an embodiment, a method is provided which involves the administrationof a dual function anti-glycPD-L1 antibody alone or in combination withanother anticancer agent to a patient in need thereof, i.e., a patientwith a cancer or tumor. Prior to administration of the anti-glycPD-L1antibody, a sample of the patient's tumor or cancer may be evaluated forthe presence of PD-L1. If the results of such an evaluation reveals thatthe patient's tumor or cancer is positive for glycosylated PD-L1, thepatient would be selected for treatment based on the likelihood thatpatient's glycPD-L1+ tumor or cancer would be more amenable to treatmentwith the anti-glycPD-L1 antibody and treatment may proceed with a morelikely beneficial outcome. A medical professional or physician mayadvise the patient to proceed with the anti-glycPD-L1 antibody treatmentmethod, and the patient may decide to proceed with treatment based onthe advice of the medical professional or physician. In addition, duringthe course of treatment, the patient's tumor or cancer cells may beassayed for the presence of glycosylated PD-L1 as a way to monitor theprogress or effectiveness of treatment. If the assay shows a change,loss, or decrease, for example, in glycosylated PD-L1 on the patient'stumor or cancer cells, a decision may be taken by the medicalprofessional in conjunction with the patient as to whether the treatmentshould continue or be altered in some fashion, e.g., a higher dosage,the addition of another anti-cancer agent or therapy, and the like.

Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe treatment or therapeutic methods of the present embodiments. Theterm “chemotherapy” refers to the use of drugs to treat cancer. A“chemotherapeutic agent” connotes a compound or composition that isadministered in the treatment of cancer. Such agents or drugs arecategorized by their mode of activity within a cell, for example,whether and at what stage they affect the cell cycle and cell growth andproliferation. Alternatively, a chemotherapeutic agent may becharacterized based on its ability to directly cross-link DNA, tointercalate into DNA, or to induce chromosomal and mitotic aberrationsby affecting nucleic acid synthesis in a cell.

Nonlimiting examples of chemotherapeutic agents include alkylatingagents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, suchas busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines,including altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gamma 1 andcalicheamicin omega 1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove.

Radiotherapy

Radiotherapy includes treatments with agents that cause DNA damage.Radiotherapy has been used extensively in cancer and disease treatmentsand embraces what are commonly known as γ-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DNAdamaging factors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA itself, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Exemplary dosage ranges for X-rays range from daily dosesof 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks) tosingle doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopesvary widely and depend on the half-life of the isotope, the strength andtype of radiation emitted, the uptake by the neoplastic cells, andtolerance of the subject undergoing treatment.

Immunotherapy

In some embodiments of the methods, immunotherapies may be used incombination or in conjunction with administration of dual functionanti-glycPD-L1 antibodies as described herein. In the context of cancertreatment, immunotherapeutics generally rely on the use of immuneeffector cells and molecules to target and destroy cancer cells.Rituximab (RITUXAN®) is such an example. Other checkpoint inhibitors canalso be administered in combination, including ipilimumab. The dualfunction anti-glycPD-L1 antibodies may also be administered incombination with other anti-PD-1 or anti-PD-L1 inhibitors, such asantibodies against PD-L1, which include atezolizumab, durvalumab, oravelumab, or antibodies against PD-1, including nivolumab,pembrolizumab, or pidilizumab. In addition, one or more of the dualfunction anti-glycPD-L1 antibodies of the embodiments may beadministered in combination with each other. The immune effector may be,for example, an antibody specific for a marker (cell surface protein orreceptor) on the surface of a tumor cell. The antibody alone may serveas an effector of therapy or it may recruit other cells to actuallyaffect cell killing. The antibody also may be conjugated to a drug ortoxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin,pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target, e.g., the PD-1 on T-cells/PD-L1 on tumor cells interaction.Various effector cells include cytotoxic T cells and natural killer (NK)cells.

In one aspect of immunotherapy, the tumor cell must bear some marker(protein/receptor) that is amenable to targeting. Optimally, the tumormarker protein/receptor is not present on the majority of other cells,such as non-cancer cells or normal cells. Many tumor markers exist andany of these may be suitable for targeting by another drug or therapyadministered with an anti-glycPD-L1 antibody in the context of thepresent embodiments. Common tumor markers include, for example, CD20,carcinoembryonic antigen (CEA), tyrosinase (p9′7), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erbB, andp155. An alternative aspect of immunotherapy is to combine anticancereffects with immune stimulatory effects. Immune stimulating moleculesalso exist and include cytokines, such as IL-2, IL-4, IL-12, GM-CSF,gamma-IFN; chemokines, such as MIP-1, MCP-1, IL-8; and growth factors,such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998, Infection Immun.,66(11):5329-5336; Christodoulides et al., 1998, Microbiology, 144(Pt11):3027-3037); cytokine therapy, e.g., α, β, and γ interferons; IL-1,GM-CSF, and TNF (Bukowski et al., 1998, Clinical Cancer Res.,4(10):2337-2347; Davidson et al., 1998, J. Immunother., 21(5):389-398;Hellstrand et al., 1998, Acta Oncologica, 37(4):347-353); gene therapy,e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998, Proc. Natl. Acad. Sci.USA, 95(24):14411-14416; Austin-Ward et al., 1998, Revista Medial deChile, 126(7):838-845; U.S. Pat. Nos. 5,830,880 and 5,846,945); andmonoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, andanti-p185 (Hollander, 2012, Front. Immun., 3:3; Hanibuchi et al., 1998,Int. J. Cancer, 78(4):480-485; U.S. Pat. No. 5,824,311). It iscontemplated that one or more anti-cancer therapies may be employed withthe antibody therapies described herein.

Surgery

Approximately 60% of individuals with cancer undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asanti-glycPD-L1 antibody treatment as described herein, chemotherapy,radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/oralternative therapies, as well as combinations thereof. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically-controlled surgery(Mohs' surgery). Upon excision of part or all of cancerous cells,tissue, or tumor, a cavity may be formed in the body. Treatment may beaccomplished by perfusion, direct injection, or local application of thearea with an additional anti-cancer therapy. Such treatment may berepeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2,3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months. These treatments may be of varying dosages as well.

Protein Purification

Protein, including antibody and, specifically, anti-glycPD-L1 antibody,purification techniques are well known to those of skill in the art.These techniques involve, at one level, the homogenization and crudefractionation of the cells, tissue, or organ into polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity) unless otherwise specified. Analytical methods particularlysuited to the preparation of a pure protein or peptide are ion-exchangechromatography, size-exclusion chromatography, reverse phasechromatography, hydroxyapatite chromatography, polyacrylamide gelelectrophoresis, affinity chromatography, immunoaffinity chromatography,and isoelectric focusing. A particularly efficient method of purifyingpeptides is fast-performance liquid chromatography (FPLC) or evenhigh-performance liquid chromatography (HPLC). As is generally known inthe art, the order of conducting the various purification steps may bechanged, and/or certain steps may be omitted, and still result in asuitable method for the preparation of a substantially purifiedpolypeptide.

A purified polypeptide, such as an anti-glycPD-L1 antibody as describedherein, refers to a polypeptide which is isolatable or isolated fromother components and purified to any degree relative to itsnaturally-obtainable state. An isolated or purified polypeptide,therefore, also refers to a polypeptide free from the environment inwhich it may naturally occur, e.g., cells, tissues, organs, biologicalsamples, and the like. Generally, “purified” will refer to a polypeptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. A “substantially purified” compositionrefers to one in which the polypeptide forms the major component of thecomposition, and as such, constitutes about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, or more of the protein component of thecomposition.

Various methods for quantifying the degree of purification ofpolypeptides, such as antibody proteins, are known to those of skill inthe art in light of the present disclosure. These include, for example,determining the specific activity of an active fraction, or assessingthe amount of polypeptides within a fraction by SDS/PAGE analysis. Apreferred method for assessing the purity of a fraction is to calculatethe specific activity of the fraction, to compare it to the specificactivity of the initial extract, and to thus calculate the degree ofpurity therein, assessed by a “fold purification number.” The actualunits used to represent the amount of activity will, of course, bedependent upon the particular assay technique chosen to follow thepurification, and whether or not the expressed polypeptide exhibits adetectable activity.

There is no general requirement that the polypeptide will always beprovided in its most purified state. Indeed, it is contemplated thatless substantially purified products may have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance (protein) to be isolated and amolecule to which it can specifically bind, e.g., a receptor-ligand typeof interaction. The column material (resin) is synthesized by covalentlycoupling one of the binding partners to an insoluble matrix. The columnmaterial is then able to specifically adsorb the substance from thesolution that is passed over the column resin. Elution occurs bychanging the conditions to those in which binding will be disrupted/willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that does not adsorb molecules to anysignificant extent and that has a broad range of chemical, physical, andthermal stability. The ligand should be coupled in such a way as to notaffect its binding properties. The ligand should also provide relativelytight binding; however, elution of the bound substance should occurwithout destroying the sample protein desired or the ligand.

Size-exclusion chromatography (SEC) is a chromatographic method in whichmolecules in solution are separated based on their size, or in moretechnical terms, their hydrodynamic volume. It is usually applied tolarge molecules or macromolecular complexes, such as proteins andindustrial polymers. Typically, when an aqueous solution is used totransport the sample through the column, the technique is known as gelfiltration chromatography, versus the name gel permeationchromatography, which is used when an organic solvent is used as amobile phase. The underlying principle of SEC is that particles ofdifferent sizes will elute (filter) through a stationary phase atdifferent rates, resulting in the separation of a solution of particlesbased on size. Provided that all of the particles are loadedsimultaneously or near simultaneously, particles of the same size shouldelute together.

High-performance (aka high-pressure) liquid chromatography (HPLC) is aform of column chromatography used frequently in biochemistry andanalytical chemistry to separate, identify, and quantify compounds. HPLCutilizes a column that holds chromatographic packing material(stationary phase), a pump that moves the mobile phase(s) through thecolumn, and a detector that shows the retention times of the molecules.Retention time varies depending on the interactions between thestationary phase, the molecules being analyzed, and the solvent(s) used.

Pharmaceutical Preparations

Where clinical application of a pharmaceutical composition containing adual function, anti-glycPD-L1 antibody or glycosylated PD-L1 polypeptideis undertaken, it is generally beneficial to prepare a pharmaceutical ortherapeutic composition appropriate for the intended application. Ingeneral, pharmaceutical compositions may comprise an effective amount ofone or more polypeptides or additional agents dissolved or dispersed ina pharmaceutically acceptable carrier. In certain embodiments,pharmaceutical compositions may comprise, for example, at least about0.1% of a polypeptide or antibody. In other embodiments, a polypeptideor antibody may comprise between about 2% to about 75% of the weight ofthe unit, or between about 25% to about 60%, for example, and any rangederivable there between, including the upper and lower values. Theamount of active compound(s) in each therapeutically useful compositionmay be prepared in such a way that a suitable dosage will be obtained inany given unit dose. Factors, such as solubility, bioavailability,biological half-life, route of administration, product shelf life, aswell as other pharmacological considerations, are contemplated by oneskilled in the art of preparing such pharmaceutical formulations, and assuch, a variety of dosages and treatment regimens may be desirable.

Further in accordance with certain aspects, the composition suitable foradministration may be provided in a pharmaceutically acceptable carrierwith or without an inert diluent. The carrier should be assimilable andinclude liquid, semi-solid, e.g., gels or pastes, or solid carriers.Examples of carriers or diluents include fats, oils, water, salinesolutions, lipids, liposomes, resins, binders, fillers, and the like, orcombinations thereof. As used herein, “pharmaceutically acceptablecarrier” includes any and all aqueous solvents (e.g., water,alcoholic/aqueous solutions, ethanol, saline solutions, parenteralvehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueoussolvents (e.g., propylene glycol, polyethylene glycol, vegetable oil,and injectable organic esters, such as ethyloleate), dispersion media,coatings (e.g., lecithin), surfactants, antioxidants, preservatives(e.g., antibacterial or antifungal agents, anti-oxidants, chelatingagents, inert gases, parabens (e.g., methylparabens, propylparabens),chlorobutanol, phenol, sorbic acid, thimerosal), isotonic agents (e.g.,sugars, sodium chloride), absorption delaying agents (e.g., aluminummonostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers,amino acids, such as glycine and lysine, carbohydrates, such asdextrose, mannose, galactose, fructose, lactose, sucrose, maltose,sorbitol, mannitol, etc), gels, binders, excipients, disintegrationagents, lubricants, sweetening agents, flavoring agents, dyes, fluid andnutrient replenishers, such like materials and combinations thereof, aswould be known to one of ordinary skill in the art. Except insofar asany conventional media, agent, diluent, or carrier is detrimental to therecipient or to the therapeutic effectiveness of the compositioncontained therein, its use in administrable composition for use inpracticing the methods is appropriate. The pH and exact concentration ofthe various components in a pharmaceutical composition are adjustedaccording to well-known parameters. In accordance with certain aspects,the composition is combined with the carrier in any convenient andpractical manner, i.e., by solution, suspension, emulsification,admixture, encapsulation, absorption, grinding, and the like. Suchprocedures are routine for those skilled in the art.

In certain embodiments, the compositions may comprise different types ofcarriers depending on whether they are to be administered in solid,liquid, or aerosol form, and whether it needs to be sterile for theroute of administration, such as injection. The compositions can beformulated for administration intravenously, intradermally,transdermally, intrathecally, intra-arterially, intraperitoneally,intranasally, intravaginally, intrarectally, intramuscularly,subcutaneously, mucosally, orally, topically, locally, by inhalation(e.g., aerosol inhalation), by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, via acatheter, via a lavage, in lipid compositions (e.g., liposomes), or byother methods or any combination of the forgoing as would be known toone of ordinary skill in the art. See, for example, RemingtonPharmaceutical Sciences, 18th Ed., 1990. Typically, such compositionscan be prepared as either liquid solutions or suspensions; solid orreconstitutable forms suitable for use to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and, the preparations can also be emulsified.

The antibodies may be formulated into a composition in a free base,neutral, or salt form. Pharmaceutically acceptable salts include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids,such as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric, or mandelic acid. Salts formed withthe free carboxyl groups may also be derived from inorganic bases, suchas, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine, or procaine.

In further embodiments, a pharmaceutical lipid vehicle composition thatincludes polypeptides, one or more lipids, and an aqueous solvent may beused. As used herein, the term “lipid” refers to any of a broad range ofsubstances that are characteristically insoluble in water andextractable with an organic solvent. This broad class of compounds iswell known to those of skill in the art, and as the term “lipid” is usedherein, it is not limited to any particular structure. Examples includecompounds that contain long-chain aliphatic hydrocarbons and theirderivatives. A lipid may be naturally occurring or synthetic (i.e.,designed or produced by man). However, a lipid is usually a biologicalsubstance. Biological lipids are well known in the art, and include forexample, neutral fats, phospholipids, phosphoglycerides, steroids,terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,lipids with ether- and ester-linked fatty acids, polymerizable lipids,and combinations thereof. Of course, compounds other than thosespecifically described herein that are understood by one of skill in theart as lipids are also encompassed by the compositions and methods. Oneof ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the antibody may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid, contained or complexed with amicelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The term “unit dose” or “dosage” refers to physically discrete unitssuitable for use in a subject, each unit containing a predeterminedquantity of the therapeutic antibody or composition containing thetherapeutic antibody calculated to produce the desired responsesdiscussed above in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the effect desired. The actual dosage amount of a compositionof the present embodiments administered to a patient or subject can bedetermined by physical and physiological factors, such as body weight,the age, health, and sex of the subject, the type of disease beingtreated, the extent of disease penetration, previous or concurrenttherapeutic interventions, idiopathy of the patient, the route ofadministration, and the potency, stability, and toxicity of theparticular therapeutic substance. In other non-limiting examples, a dosemay also comprise from about 1 microgram/kg/body weight, about 5microgram/kg/body weight, about 10 microgram/kg/body weight, about 50microgram/kg/body weight, about 100 microgram/kg/body weight, about 200microgram/kg/body weight, about 350 microgram/kg/body weight, about 500microgram/kg/body weight, about 1 milligram/kg/body weight, about 5milligram/kg/body weight, about 10 milligram/kg/body weight, about 50milligram/kg/body weight, about 100 milligram/kg/body weight, about 200milligram/kg/body weight, about 350 milligram/kg/body weight, about 500milligram/kg/body weight, to about 1000 milligram/kg/body weight or moreper administration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 5 milligram/kg/body weight to about 100 milligram/kg/body weight,about 5 microgram/kg/body weight to about 500 milligram/kg/body weight,etc., can be administered, based on the numbers described above. Theforegoing doses include amounts between those indicated and are intendedto also include the lower and upper values of the ranges. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject.

The particular nature of the therapeutic composition or preparation isnot intended to be limiting. For example, suitable compositions may beprovided in formulations together with physiologically tolerable liquid,gel, or solid carriers, diluents, and excipients. In some embodiments,the therapeutic preparations may be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual subjects, as described supra.

Glycosylated PD-L1 as a Biomarker

Provided are methods involving the use of at least one dual functionanti-glycPD-L1 antibody in assays for glycosylated PD-L1 as a biomarker.Such methods may be useful in biomarker evaluations of the tumor orcancer cells obtained from a subject who has a cancer or tumor. Providedis a method to determine whether a subject who has cancer also has acancer or tumor that expresses glycosylated PD-L1, particularly, adetectable level of glycosylated PD-L1 on the cell surface of suchcells. For example, if the subject's cancer or tumor cells are testedand determined to express glycosylated PD-L1 on the cell surface, thenit is more likely that the subject treated with an anti-glycPD-L1antibody as described, alone, or in combination with another anti-canceragent, for example, would benefit from the treatment. Such methodscomprise obtaining a sample from a subject having a cancer or tumor,testing the sample for the presence of glycosylated PD-L1 on cellsderived from the subject's cancer or tumor using binding methods knownand used in the art, or as described supra, and administering to thesubject an effective amount of an anti-glycPD-L1 antibody alone, or incombination with another anti-cancer agent, if the subject's cancer ortumor is found to be positive for the cell surface expression ofglycosylated PD-L1 protein. Diagnosing the subject as having a cancer ortumor expressing glycosylated PD-L1 prior to treatment allows for moreeffective treatment and benefit to the subject, as the administeredanti-glycPD-L1 antibody is more likely to block or inhibit theinteraction of the subject's glycosylated PD-L1-expressing cancer ortumor cells with the subject's PD-1-expressing T-cells, therebypreventing immunosuppression of the T-cell activity and promotingkilling of the tumor or cancer cells by activated T-cell killing. In anembodiment, the method may involve first selecting a subject whosecancer or tumor may be amenable to testing for the presence of expressedglycosylated PD-L1 protein.

Similar methods may be used to monitor the presence of glycosylatedPD-L1 on a patient's tumor cells during a course of cancer treatment ortherapy, including combination treatments with an anti-glycPD-L1antibody and another anticancer drug or treatment, over time, as well asafter treatment has ceased. Such methods may also be used in companiondiagnostic methods in which an anti-cancer treatment regimen, orcombination treatment, involves testing or assaying a patient's tumor orcancer sample for glycosylated PD-L1-expressing tumor or cancer cells,prior to treatment and during the course of treatment, e.g., monitoring,to determine a successful outcome or the likelihood thereof.

In other embodiments, the anti-glycPD-L1 antibodies may be conjugated toa marker useful for in vivo imaging, for example, a radionuclide such as1¹²⁴ for PET or SPECT, a fluorochrome for optical imaging, or aparamagnetic chelate for MM. The labeled anti-glycPD-L1 antibodies canbe administered to the patient, for example, intravenous or otherparental mode of administration, and, after waiting the appropriateamount of time, detecting the presence and location of the labeledantibodies to localize and quantitate the tumor cells.

Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions mayincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents may be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

Kits and Diagnostics

In another embodiment, a kit containing therapeutic agents and/or othertherapeutic and delivery agents is provided. In some embodiments, thekit is used for preparing and/or administering a therapy involving theanti-glycPD-L1 antibodies described herein. The kit may comprise one ormore sealed vials containing any of the pharmaceutical compositions asdescribed herein. The kit may include, for example, at least oneanti-glycosylated PD-L1 antibody, as well as reagents to prepare,formulate, and/or administer one or more anti-glycPD-L 1 antibodies orto perform one or more steps of the described methods. In someembodiments, the kit may also comprise a suitable container means, whichis a container that will not react with components of the kit, such asan Eppendorf tube, an assay plate, a syringe, a bottle, or a tube. Thecontainer may be made from sterilizable materials, such as plastic orglass.

The kit may further include an instruction sheet that outlines theprocedural steps of the methods set forth herein, and will followsubstantially the same procedures as described herein or are known tothose of ordinary skill. The instruction information may be in acomputer readable medium containing machine-readable instructions that,when executed using a computer, cause the display of a real or virtualprocedure of delivering a pharmaceutically effective amount of thetherapeutic agent.

Fusions and Conjugates

The anti-glycosylated PD-L1 antibodies or glycosylated PD-L1polypeptides provided herein can also be expressed as fusion proteinswith other proteins or chemically conjugated to another moiety. In someembodiments, the antibodies or polypeptides have an Fc portion that canbe varied by isotype or subclass, can be a chimeric or hybrid, and/orcan be modified, for example to improve effector functions, controlhalf-life or tissue accessibility, augment biophysical characteristics,such as stability, and improve efficiency of production, which can beassociated with cost reductions. Many modifications useful in theconstruction of fusion proteins and methods for making them are known inthe art, for example, as reported by Mueller, J. P. et al., 1997, Mol.Immun. 34(6):441-452; Swann, P. G., 2008, Curr. Opin. Immunol.,20:493-499; and Presta, L. G., 2008, Curr. Opin. Immunol., 20:460-470.In some embodiments, the Fc region is the native IgG1, IgG2, or IgG4 Fcregion of the antibody. In some embodiments, the Fc region is a hybrid,for example a chimera containing IgG2/IgG4 Fc constant regions.Modifications to the Fc region include, but are not limited to, IgG4modified to prevent binding to Fc gamma receptors and complement; IgG1modified to improve binding to one or more Fc gamma receptors; IgG1modified to minimize effector function (amino acid changes); IgG1 withaltered/no glycan (typically by changing expression host); and IgG1 withaltered pH-dependent binding to FcRn. The Fc region can include theentire hinge region, or less than the entire hinge region of theantibody.

Another embodiment includes IgG2-4 hybrids and IgG4 mutants that havereduced binding to FcR which increase their half-life. RepresentativeIG2-4 hybrids and IgG4 mutants are described, for example, in Angal etal., 1993, Molec. Immunol., 30(1):105-108; Mueller et al., 1997, Mol.Immun., 34(6):441-452; and U.S. Pat. No. 6,982,323; all of which arehereby incorporated by references in their entireties. In someembodiments, the IgG1 and/or IgG2 domain is deleted. For example, Angalet al., Id., describe proteins in which IgG1 and IgG2 domains haveserine 241 replaced with a proline. In some embodiments, fusion proteinsor polypeptides having at least 10, at least 20, at least 30, at least40, at least 50, at least 60, at least 70, at least 80, at least 90 orat least 100 amino acids are contemplated.

In some embodiments, anti-glycosylated PD-L1 antibodies or glycosylatedPD-L1 polypeptides are linked to or covalently bind or form a complexwith at least one moiety. Such a moiety may be, but is not limited to,one that increases the efficacy of the antibody as a diagnostic or atherapeutic agent. In some embodiments, the moiety can be an imagingagent, a toxin, a therapeutic enzyme, an antibiotic, a radio-labelednucleotide, a chemotherapeutic agent, and the like.

In some embodiments, the moiety that is conjugated or fused to ananti-glycPD-L1 antibody or glycosylated polypeptide or portion thereofmay be an enzyme, a hormone, a cell surface receptor, a toxin, such as,without limitation, abrin, ricin A, pseudomonas exotoxin (i.e., PE-40),diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein), aprotein (such as tumor necrosis factor, interferon (e.g., α-interferon,(3-interferon), nerve growth factor (NGF), platelet derived growthfactor (PDGF), tissue plasminogen activator (TPA), or an apoptotic agent(e.g., tumor necrosis factor-α, tumor necrosis factor-β)), a biologicalresponse modifier (such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”)),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or macrophage colony stimulatingfactor, (“M-CSF”), or growth factors (e.g., growth hormone (“GH”)), acytotoxin (e.g., a cytostatic or cytocidal agent, such as paclitaxel,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, a tubulysin-basedmicrotubule inhibitor, e.g., a Maytansinoid, such as Maytansinoid DM1(N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine; mertansine oremtansine, depending on the linker used; and Maytansinoid DM4(N2′-deacetyl-n2′-(4-Mercapto-4-Methyl-1-oxopentyl)-6-MethylMaytansine;ravtansine), ImmunoGen, Inc., Waltham, Mass.) and puromycin and analogsor homologs thereof), antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, BiCNU® (carmustine; BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C,and cisdichlorodiamine platinum (II) (DDP) cisplatin), an anthracycline(e.g., daunorubicin (formerly daunomycin) and doxorubicin), anantibiotic (e.g., dactinomycin (formerly actinomycin), bleomycin,mithramycin, and anthramycin (AMC)), an anti-mitotic agent and/ortubulin inhibitor e.g., vincristine and vinblastine, monomethylauristatin F (MMAF), monomethyl auristatin E (or desmethyl-auristatin E)(MMAE), e.g., vedotin; or combinations thereof.

Antibody-Drug Conjugates (ADCs)

Antibody drug conjugates (ADCs) are biologic therapeutic agents in whichpotent cytotoxic drugs are covalently linked via chemical linkers orcoupling agents to antibodies, typically monoclonal antibodies, whichare directed to specific target antigens, in particular, target antigensexpressed or overexpressed on the surfaces of tumor or cancer cells.Such “loaded” antibodies are designed to deliver lethal cytotoxiccargoes to tumor or cancer cells. ADCs provide a means for targeting thepayload drug to neoplastic cells while reducing side effects andminimizing systemic toxicity. ADCs bind to the cell surface-expressedtarget antigen by virtue of the specific interaction of the antibodycomponent of the ADC and its target antigen. After binding to the targetantigen, the ADC may be internalized into the cell, particularly, if theantibody has heightened internalization activity, as do theanti-glycPD-L1 antibodies of the embodiments described herein, e.g.,STM108 and STM073. Accordingly, when such ADCs are internalized into thecell, they act directly to kill the cell or target a molecule inside thecell, which leads to apoptosis or cell death. Such ADCs comprising theanti-glycPD-L1 antibodies described herein, particularly, monoclonal,humanized, chimeric, or human antibodies, combine the specific targetingof antibodies to glycosylated PD-L1 on tumor and cancer cells with thecancer-killing ability of cytotoxic drugs or compounds, therebyproviding further advantages for treatment and therapies with theanti-glycPD-L1 antibodies. Techniques for preparing and using ADCs areknown in the art and are not intended to be limiting for theanti-glycPD-L1 antibodies described herein. (See, e.g., ValliereDouglass, J. F., et al., 2015, Mol. Pharm., 12(6):1774-1783; Leal, M. etal., 2014, Ann. N.Y. Acad. Sci., 1321:41-54; Panowski, S. et al., 2014,mAbs, 6(1):34-45; Beck, A. 2014, mAbs, 6(1):30-33; Behrens, C. R. etal., 2014, mAbs, 6(1):46-53; and Flygare, J. A. et al., 2013, Chem.Biol. Drug Des., 81(1):113-121). In embodiments, some or all of theabove-described moieties, particularly, toxins and cytotoxins, may beconjugated to an anti-glycPD-L1 antibody to produce effective ADCs fortreating cancer. In embodiments, the anti-glycPD-L1 antibody componentof the ADC may be a bispecific, multispecific, biparatopic, ormultiparatopic antibody.

Techniques for conjugating therapeutic or cytotoxic moieties toantibodies are well known; See, e.g., Amon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMONOCLONAL ANTIBODIES AND CANCER THERAPY, Reisfeld et al. (eds.), 1985,pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies For DrugDelivery”, in CONTROLLED DRUG DELIVERY (2nd Ed.), Robinson et al.(eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MONOCLONALANTIBODIES '84: BIOLOGICAL AND CLINICAL APPLICATIONS, Pinchera et al.(eds.), 1985, pp. 475-506); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMONOCLONAL ANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al.(eds.), 1985, pp. 303-16, Academic Press; Thorpe et al., Immunol. Rev.62:119-158 (1982); Carter et al., Cancer J. 14(3):154-169 (2008); Alleyet al., Curr. Opin. Chem. Biol. 14(4):529-537 (2010); Carter et al.,Amer. Assoc. Cancer Res. Educ. Book. 2005(1):147-154 (2005); Carter etal., Cancer J. 14(3):154-169(2008); Chari, Acc. Chem Res. 41(1):98-107(2008); Doronina et al., Nat. Biotechnol. 21(7):778-784(2003); Ducry etal., Bioconjug Chem. 21(1):5-13(2010); Senter, Curr Opin. Chem. Biol.13(3):235-244 (2009); and Teicher, Curr Cancer Drug Targets.9(8):982-1004 (2009). A review of ADCs and ADC oncology products isfound in Lambert, British J. Clin. Pharmacol., 76(2):248-262 (2013) andin Bouchard et al., Bioorganic & Medicinal Chemistry Letters,24:5357-5363 (2014).

In specific embodiments, the anti-glycPD-L1 antibodies, which facilitatethe internalization of PD-L1 into tumor cells, are conjugated to ahighly potent biologically active drug or agent, such as a cytotoxicand/or chemotherapeutic agent, a toxin or cytotoxin as noted above, or aradionuclide, typically by chemical linkers with labile bonds, toproduce an anti-glycPD-L1 antibody-drug conjugate (ADC), called ananti-glycPD-L1 antibody-ADC herein. The biologically active drug orcytotoxic agent, for example, serves as a “cytotoxic payload,” which isdelivered into a cell, particularly a tumor or cancer cell expressing acell-surface target receptor or molecule that is bound by theanti-glycPD-L1 antibody-ADC. Such an anti-glycPD-L 1 antibody-ADC boundto its target molecule is internalized into the cell where the cytotoxicdrug payload is released. Enhancement of the cancer cell-killingactivity of the internalizing anti-glycPD-L1 antibodies described hereinthrough conjugation to highly potent cytotoxic payloads affordsanti-cancer ADC biologics having high anti-tumor activity and generallymild adverse effects that are well-tolerated.

An anti-glycPD-L1 antibody as described in the embodiments herein may belinked to various types of cytotoxic or DNA-acting payloads as known andused in the art, or as yet to be commercialized. For example, maytansineis a benzoansamacrolide that was first isolated from the bark of theEthiopian shrub Maytenus ovutus. This cytotoxic agent and derivativesthereof (e.g., maytansinoids) bind to tubulin near the Vinca alkaloidbinding site. They are considered to have a high affinity for tubulinlocated at the ends of microtubules and lower affinity to sitesdistributed throughout the microtubules. The suppression of microtubuledynamics causes cells to arrest in the G2/M phase of the cell cycle,ultimately resulting in cell death by apoptosis. (Oroudjev et al., Mol.Cancer Ther., 10L2700-2713 (2010)). Two maytansine derivatives(thiol-containing maytansinoids) include DM1 and DM4 (ImmunoGen, Inc.,Waltham, Mass.) have been widely used in combination with irreversibleand reversible linkers. In particular, DM1 attached to an antibody witha thioether linker is called “emtansine;” DM1 attached to an antibodywith an SPP linker is called “mertansine;”. DM4 attached with an SPDBlinker is called “ravtansine;” and DM4 attached with an sSPDB linker iscalled “soravtansine.” (ImmunoGen, Inc., Waltham, Mass.). In anembodiment, the anti-glycPD-L1 antibody-ADC comprises the tubulin-actingmaytansinoid payload DM1. In an embodiment, the anti-glycPD-L1antibody-ADC comprises the tubulin-acting maytansinoid payload DM4. Inan embodiment, the anti-glycPD-L1 antibody-ADC comprises a DNA-actingpayload, e.g., DGN462 (ImmunoGen, Inc., Waltham, Mass.). In anembodiment, the anti-glycPD-L1 antibody component of the anti-glycPD-L1antibody-ADC is STM073, or a binding portion thereof. In an embodiment,the anti-glycPD-L1 antibody component of the anti-glycPD-L1 antibody-ADCis STM108, or a binding portion thereof.

In a particular embodiment, the cytotoxic agent conjugated to theanti-glycPD-L1 antibody is MMAE (monomethyl auristatin E (ordesmethyl-auristatin E)), a highly toxic, antineoplastic agent whoseantimitotic activity involves inhibiting cell division by blocking thepolymerization of tubulin. Vedotin, an International NonproprietaryName, refers to MMAE plus its linking structure to an antibody in anMMAE-antibody conjugate. In more particular embodiments, the ADC isSTM073-MMAE or STM108-MMAE.

A number of chemical linkers are known and used for conjugating acytotoxic or DNA-acting drug payload to an antibody to produce ADCs.Certain linkers embraced for use alone or in combination for producingADCs comprising the anti-glycPD-L1 antibodies, particularly, those thatinternalize after binding their target as described herein, include SMCC(4-(N-Maleimidomethyl) cyclohexanecarboxylic acid N-hydroxysuccinimideester); SPDB (N-succinimidyl 3-(2-pyridyldithio)butyrate); SPP(N-succinimidyl 4-(2-pyridyldithio)pentanoate); sulfo-SPDB or sSPDB(N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate); the thioetherlinker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(MCC); and vc (valine-citrulline dipeptide linker). By way of example,engineered linkers (e.g., SMCC, SPDB, S-SPDB), (Immunogen, Inc.) havebeen designed to be stable prior to the binding of an ADC to a tumor andthen to optimize payload efficacy once the ACD is internalized inside acancer cell. Other linkers, such as the dipeptide vc linker, which is acathepsin-cleavable linker, may be used to conjugate an antibody to acytotoxic agent, such as an auristatin which is a mitotic inhibitorderived from dolastatin 10, e.g., monomethylauristatin E (MMAE), e.g.,vedotin. The cytotoxins may be conjugated to the antibody such that morethan one toxin molecule is attached to each antibody molecule, forexample, there may be, on average, 2, 3, 4, 5, 6, 7 or 8 toxin moleculesper antibody.

In a particular embodiment, MMAE is indirectly linked to antibodycysteines by a maleimidocaproyl (MC) attachment group, which is coupledto valine-citrulline-p-aminobenzyloxycarbonyl-MMAE (MC-vc-PAB-MMAE). Inthe “MC-vc-PAB-MMAE” linear structure, “MC” consists of maleimide andcaproic acid and is the moiety that attaches to an antibody, typicallyvia cysteine groups on the H chain. In turn, “MC” is attached to a “vc”linker which consists of valine (Val) and citruline (Cit) and which is acathepsin-cleavable linker that is cleaved by cathepsin inside of tumoror cancer cells. “vc” is attached to the spacer “PAB”, i.e.,paraminobenzoic acid, to which the MMAE cytotoxin is linked.MC-vc-PAB-MMAE ADCs release free, membrane-permeable MMAE when cleavedby proteases such as cathepsin B. In an embodiment, the linker to theantibody is stable in extracellular fluid, but is cleaved by cathepsinonce the ADC has entered a tumor or cancer cell, thus activating theantimitotic mechanism of MMAE or other toxin drug. In anotherembodiment, monomethylauristatin F, (MMAF) is linked to antibodycysteines by maleimidocaproyl (MC-MMAF). In contrast to MC-vc-PAB-MMAEADCs, MC-MMAF ADCs are uncleavable, like MCC-DM1 ADCs, and must beinternalized and degraded within a cell, releasing cysteine-MC-MMAF asthe active drug inside the cell.

In an embodiment, the cytotoxic payload is released in the lysosomefollowing internalization of the ADC into a cell. In the lysosome,lysosomal enzymes digest the antibody component of the ADC. Followinglysosomal degradation, the drug (and drug-linker) payload is releasedinto the cytoplasm, where the drug binds intracellular targets,ultimately causing cell death. Optimally, the released payload is fullyactive, with the linker still attached. In other embodiments in whichthe target bound to the ADC results in poor trafficking to the lysosome,linkers which are stable outside of the target cell, but which cleavethe payload from the antibody component once inside the cell provide analternative mode for payload release within the cell, but outside of thelysosome. In other embodiments, the linker is stable in extracellularfluid, but is cleaved by cathepsin once the ADC has entered a tumor orcancer cell, thus activating the antimitotic or other cytotoxicmechanism of the toxin drug. In other embodiments, a payload released bythe action of cleavable linkers is able to enter a neighboring cancercells and kill them via a bystander effect, thus augmenting thetargeting and tumor killing activity of an ADC.

In an embodiment, an anti-glycPD-L1 antibody as described herein, suchas dual function STM073 or STM108 MAbs, is coupled to DM1 via the linkerSMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Inanother embodiment, an anti-glycPD-L1 antibody as described herein, suchas STM073 or STM108, is coupled to DM4 via the linker SPDB(N-succinimidyl 3-(2-pyridyldithio)butyrate) or sSPDB(N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate). In anotherembodiment, the auristatin monomethylauristatin E is linked to cysteineresidues of an anti-glycPD-L1 antibody as described herein, such asSTM073 or STM108, bymaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl(MC-vc-PAB-MMAE). In a particular embodiment, the ADC isSTM108-MC-vc-PAB-MMAE. In another particular embodiment, the ADC isSTM073-MC-vc-PAB-MMAE. In an embodiment, the anti-glycPD-L1 antibody-ADCcomprises multiple units of a drug, such as MMAE, per molecule, such as1-10, 1-5, 2-5, 3-5, or 2, 3, 4, 5, 6, 7, 8, 9, 10 units of drug, suchas MMAE, per molecule, as well as values therebetween. In otherembodiments, the antibody-drug ratio may be 2, 3, 4, 5, 6, 7, 8, 9, 10,or greater, as well as ranges between 2-10 and values therebetween. Inembodiments, the STM108 or STM073 antibody is a bispecific,multispecific, biparatopic, or multiparatopic antibody, or an antigenbinding portion thereof. Such ADCs comprising a dual functionanti-glycPD-L1 antibody as described herein, e.g., the above-mentionedSTM108-MC-vc-PAB-MMAE, provide bolstered and multifaceted antineoplasticeffects in the killing of tumor and cancer cells for cancer treatment.As but a few illustrative advantages, an anti-human glycPD-L1 antibody(e.g., MAb)-ADC, e.g., STM108-ADC, can block the PD-1/PD-L1 interaction,thereby enhancing T cell immunity and effector function against tumorcells; it can selectively target glycosylated PD-L1 expressed on tumorand cancer cells; it can internalize PD-L1 on tumor or cancer cellsafter binding, thereby reducing the surface-expressed PD-L1 on tumor orcancer cells and further reducing the oncogenic potential of PD-L1; itcan cause apoptosis of tumor or cancer cells into which antibody isinternalized and the toxic drug is released to damage and ultimatelykill the cell; and it can facilitate a bystander effect by killingnearby or neighboring tumor or cancer cells through the release of toxicdrug from the apoptosed tumor or cells.

In some embodiments, antibodies as described herein may be conjugated toa marker, such as a peptide, to facilitate purification. In someembodiments, the marker is a hexa-histidine peptide (SEQ ID NO: 93),i.e., the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson, I. A. et al.,Cell, 37:767-778 (1984)), or the “flag” tag (Knappik, A. et al.,Biotechniques 17(4):754-761 (1994)).

In other embodiments, the moiety conjugated to the antibodies andpolypeptides as described herein may be an imaging agent that can bedetected in an assay. Such imaging agents may be enzymes, prostheticgroups, radiolabels, nonradioactive paramagnetic metal ions, haptens,fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, bioluminescentmolecules, photoaffinity molecules, or colored particles or ligands,such as biotin. In embodiments, suitable enzymes include, but are notlimited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic group complexesinclude, but are not limited to, streptavidin/biotin and avidin/biotin;fluorescent materials include, but are not limited to, umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin;luminescent materials include, but are not limited to, luminol;bioluminescent materials include, but are not limited to, luciferase,luciferin, and aequorin; radioactive materials include, but are notlimited to, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt(⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu),manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorous(³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re),rhodium (¹⁵⁰Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc),selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc),thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions.

The imaging agent may be conjugated to the antibodies or polypeptidesdescribed herein either directly or indirectly through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 which reports onmetal ions that can be conjugated to antibodies and other molecules asdescribed herein for use as diagnostics. Some conjugation methodsinvolve the use of a metal chelate complex employing, for example, anorganic chelating agent, such as diethylenetriaminepentaacetic acidanhydride (DTPA); ethylenetriaminetetraacetic acid;N-chloro-p-toluenesulfonamide; and/ortetrachloro-3-6α-diphenylglycouril-3, attached to the antibody.Monoclonal antibodies can also be reacted with an enzyme in the presenceof a coupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers can be prepared in the presence of these couplingagents or by reaction with an isothiocyanate.

In some embodiments, the anti-glycPD-L1 antibodies or glycPD-L1polypeptides as described herein may be conjugated to a second antibodyto form an antibody heteroconjugate, for example, as described in U.S.Pat. No. 4,676,980. Such heteroconjugate antibodies can additionallybind to haptens (e.g., fluorescein), or to cellular markers (e.g.,without limitation, 4-1-BB, B7-H4, CD4, CD8, CD14, CD25, CD27, CD40,CD68, CD163, CTLA4, GITR, LAG-3, OX40, TIM3, TIM4, TLR2, LIGHT, ICOS,B7-H3, B7-H7, B7-H7CR, CD70, CD47) or to cytokines (e.g., IL-7, IL-15,IL-12, IL-4 TGF-beta, IL-10, IL-17, IFNγ, Flt3, BLys) or chemokines(e.g., CCL21).

In some embodiments, the anti-glycosylated PD-L1 antibodies orglycosylated PD-1 polypeptides described herein can also be attached tosolid supports, which can be useful for carrying out immunoassays orpurification of the target antigen or of other molecules that arecapable of binding to the target antigen that has been immobilized tothe support via binding to an antibody or antigen binding fragment asdescribed herein. Such solid supports include, but are not limited to,glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene.

EXAMPLES

The following examples are included to demonstrate embodiments thatrelate to the dual function anti-glycPD-L1 antibodies thatpreferentially bind glycosylated PD-L1 compared with unglycosylatedPD-L1 and/or PD-L1 glycosylation mutants and methods of use describedherein. Representative dual function anti-glycPD-L1 antibodies areexemplified. It should be appreciated by those of skill in the art thatthe disclosed dual function anti-glycPD-L1 antibodies are examples andare not intended to be limiting.

Example 1 Materials and Methods

Cell Culture, Stable Transfectants, and Transfection.

All cells were obtained from American Type Culture Collection (ATCC).These cells were grown in in DMEM/F12 or RPMI 1640 medium supplementedwith 10% fetal bovine serum (FBS). PD-L1 stable transfectants inMDA-MB-468, BT549 and 293T cells were selected using puromycin(InvivoGen, San Diego, Calif., USA). For transient transfection, cellswere transiently transfected with DNA, such as DNA encoding PD-L1, usingSN liposomes (Hu, M. C. et al., 2004, Cell, 117:225-237) andLipofectamine™ 2000 (Life Technologies, Carlsbad, Calif., USA).

Generation of Stable Cells Using Lentiviral Infection.

The lentiviral-based shRNA (pGIPZ plasmids) used to knockdown expressionof PD-L1 (Shen, J. et al., 2013, Nature, 497:383-387) in cells waspurchased from the shRNA/ORF Core Facility (UT MD Anderson CancerCenter). Based on knock-down efficiency of PD-L1 protein expression inMDA-MB-231 or A431 cells, the inventors selected two shPD-L1 clones forthis study. The mature antisense sequences are as follows:TCAATTGTCATATTGCTAC (shPD-L1 #1, SEQ ID NO: 34), TTGACTCCATCTTTCTTCA(shPD-L1 #5, SEQ ID NO: 35). Using a pGIPZ-shPD-L1/Flag-PD-L1 dualexpression construct to knock down endogenous PD-L1 and reconstituteFlag-PD-L1 simultaneously, the inventors established endogenous PD-L1knock-down and Flag-PD-L1 WT or 4NQ mutant expressing cell lines. Togenerate lentivirus-expressing shRNA for PD-L1 and Flag-PD-L1, theinventors transfected 293T cells with pGIPZ-non-silence (for vectorcontrol virus), pGIPZ-shPD-L1, or pGIPZ-shPD-L1/PD-L1 WT, orpGIPZ-shPD-L1/PD-L1 4NQ mutant with FuGENE 6 transfection reagent.Twenty-four hours after transfection the medium was changed, and thenthe medium was collected at 24-hour intervals. The collected mediumcontaining lentivirus was centrifuged to eliminate cell debris, andfiltered through 0.45-μm filters. Cells were seeded at 50% confluence 12hours before infection, and the medium was replaced with mediumcontaining lentivirus. After infection for 24 hours, the medium wasreplaced with fresh medium and the infected cells were selected with 1μg/ml puromycin (InvivoGen).

Plasmids.

A human PD-L1 clone was obtained from the shRNA/ORF Core Facility (UT MDAnderson Cancer Center, Houston, Tex., USA) and cloned into pCDHlentiviral expression vectors to establish PD-L1-Flag or PD-L1-Mycexpression cell lines using known molecular biological techniques. Inaddition, human PD-L1 nucleic acid was also cloned into pEGFP-N1 andpCMV-HA mammalian cell expression vectors for transient transfection.pCDH/PD-L1-Flag expression vector was used as a template to generate thePD-L1-Flag NQ mutants N35Q, N192Q, N200Q, N219Q, and 4NQ(N35Q/N192Q/N200Q/N219Q) by performing site directed mutagenesis usingprimers presented in Table 4 below. To create a pGIPZ-shPD-L1/Flag-PD-L1dual expression construct to knock down endogenous PD-L1 andreconstitute Flag-PD-L1 simultaneously, a shPD-L1 construct (shPD-L1 #5)which targets the 3»-UTR region of PD-L1 mRNA was selected. TheFlag-PD-L1 wild type (WT) or 4NQ mutant DNA was cloned intopGIPZ-shPD-L1 (Thermo Scientific, Pittsburgh, Pa., USA) which expressesshRNA specific for endogenous PD-L1. All constructs were confirmed usingenzyme digestion and DNA sequencing.

TABLE 4 Primers for site directed mutagenesis Primers Sequences (5′to 3′) N35Q Forward (SEQ ID NO: 36)gtggtagagtatggtagccaaatgacaattgaatgcaaa Reverse (SEQ ID NO: 37)tttgcattcaattgtcatttggctaccatactctaccac N192Q Forward (SEQ ID NO: 38)gagaggagaagcttttccaggtgaccagcacactgag Reverse (SEQ ID NO: 39)ctcagtgtgctggtcacctggaaaagcttctcctctc N200Q Forward (SEQ ID NO: 40)gaccagcacactgagaatccagacaacaactaatgagat Reverse (SEQ ID NO: 41)atctcattagttgttgtctggattctcagtgtgctggtc N219Q Forward (SEQ ID NO: 42)gagagaggagaagcttttccaagtgaccagcacactgaga Reverse (SEQ ID NO: 43)tctcagtgtgctggtcacttggaaaagcttctcctctctc

qRT-PCR assays were performed to measure the expression of mRNA (Shen etal., 2013, Nature, 497:383-7; and Chang et al., 2011, Nature CellBiology, 13:317-23) (see Table 5 below). Cells were washed twice withPBS and immediately lysed in QIAzol. The lysed sample was subjected tototal RNA extraction using RNeasy Mini Kit (Qiagen, Hilden, Germany). Tomeasure the expression of mRNA, cDNA was synthesized from 1 μg purifiedtotal RNA by SuperScript III First-Strand cDNA synthesis system usingrandom hexamers (Life Technologies) according to the manufacturer'sinstructions. qPCR was performed using a real-time PCR machine (iQ5,BioRad, Hercules, Calif., USA). All the data analysis was performedusing the comparative Ct method. Results were first normalized tointernal control β-actin mRNA.

TABLE 5 Primers for qRT-PCR. Gene Sequences (5′ to 3′) B4GALT2Forward (SEQ ID NO: 44) gcataacgaacctaaccctcag Reverse (SEQ ID NO: 45)gcccaatgtccactgtgata B4GALT3 Forward (SEQ ID NO: 46) gtaacctcagtcacctgccReverse (SEQ ID NO: 47) attccgctccacaatctctg B3GNT3Forward (SEQ ID NO: 48) tcttcaacctcacgctcaag Reverse (SEQ ID NO: 49)gtgtgcaaagacgtcatcatc B3GAT1 Forward (SEQ ID NO: 50)caccatcaccctcctttctattc Reverse (SEQ ID NO: 51) gaacaacaggtctgggatttctB3GAT2 Forward (SEQ ID NO: 52) gccttttgccatcgacatgReverse (SEQ ID NO: 53) agtcagattcttgcatccctg ST6GAL1Forward (SEQ ID NO: 54) caaggagagcattaggaccaag Reverse (SEQ ID NO: 55)ccccattaaacctcaggactg ST3GAL4 Forward (SEQ ID NO: 56)tcgtcatggtgtggtattcc Reverse (SEQ ID NO: 57) caggaagatgggctgatcc MAN2A2Forward (SEQ ID NO: 58) gaccgcactcatcttacacc Reverse (SEQ ID NO: 59)ggaggttggctgaaggaatac MAN2B1 Forward (SEQ ID NO: 60) tcccctgctttaaccatcgReverse (SEQ ID NO: 61) ttgtcacctatactggcgttg UGGT1Forward (SEQ ID NO: 62) ctgagtgatggaacgagtgag Reverse (SEQ ID NO: 63)tagagatgaccagatgcaacg MGAT3 Forward (SEQ ID NO: 64)gagtccaacttcacggcttat Reverse (SEQ ID NO: 65) agtggtccaggaagacatagaMGAT5 Forward (SEQ ID NO: 66) tgtgagggaaagatcaagtggReverse (SEQ ID NO: 67) gctctccaaggtaaatgaggac MOGSForward (SEQ ID NO: 68) ccactgagttcgtcaagagg Reverse (SEQ ID NO: 69)acttccttgccatctgtcac GNPTAB Forward (SEQ ID NO: 70) tggctcgctgataagttctgReverse (SEQ ID NO: 71) gtgagtctggtttgggagaag ACTBForward (SEQ ID NO: 72) gcaaagacctgtacgccaaca Reverse (SEQ ID NO: 73)tgcatcctgtcggcaatg

Antibodies and Chemicals.

The following antibodies were used in the experiments described in theExamples: Flag (F3165; Sigma-Aldrich, St. Louis, Mo., USA); Myc(11667203001; Roche Diagnostics, Indianapolis, Ind., USA); HA(11666606001; Roche Diagnostics); PD-L1 (13684; Cell SignalingTechnology, Danvers, Mass., USA); PD-L1 (329702; BioLegend, San Diego,Calif., USA,); PD-L1 (GTX117446; GeneTex, Irvine, Calif., USA); PD-L1(AF156; R&D Systems, Minneapolis, Minn., USA); PD-1 (ab52587; Abcam,Cambridge, Mass., USA); B3GNT3 (ab190458; Abcam); B3GNT3 (18098-1-AP;Proteintech, Chicago, Ill., USA); Granzyme B (ab4059; Abcam); EGFR(4267; Cell Signaling Technology); α-Tubulin (B-5-1-2; Sigma-Aldrich);and β-Actin (A2228; Sigma-Aldrich). For use in the experiments,epidermal growth factor (EGF), cycloheximide, and tunicamycin werepurchased from Sigma-Aldrich. Gefitinib, erlotinib, lapatinib,cetuximab, and AG1478 were obtained from Calbiochem Corp (Billerica,Mass., USA).

Immunoblot Analysis, Immunocytochemistry and Immunoprecipitation.

Immunoblot analysis was performed as described previously (Lim et al.,2008, Gastroenterology, 135:2128-40; and Lee et al., 2007, Cell,130:440-455). Image acquisition and quantification of band intensitywere performed using an Odyssey® infrared imaging system (LI-CORBiosciences, Lincoln, Nebr., USA). For immunoprecipitation, the cellswere lysed in buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mMethylenediaminetetraacetic acid (EDTA) and 0.5% Nonidet P-40 (NP-40))and centrifuged at 16,000×g for 30 minutes to remove debris. Clearedlysates were subjected to immunoprecipitation with antibodies. Forimmunocytochemistry, cells were fixed in 4% paraformaldehyde at roomtemperature for 15 minutes, permeabilized in 5% Triton X-100 for 5minutes, and then were stained using primary antibodies. The secondaryantibodies used were anti-mouse Alexa Fluor 488 or 594 dye conjugateand/or anti-rabbit Alexa Fluor 488 or 594 dye conjugate (LifeTechnologies). Nuclei were stained with 4′,6-diamidino-2-phenylindole(DAPI blue) (Life Technologies). After mounting, the cells werevisualized using a multiphoton confocal laser-scanning microscope (CarlZeiss, Thornwood, N.Y., USA).

PD-L1 and PD-1 (PD-L1/PD-1) Interaction Assay.

To measure the interaction of PD-1 protein and PD-L1 protein, cells werefixed in 4% paraformaldehyde at room temperature for 15 minutes and thenwere incubated with recombinant human PD-1-Fc chimera protein (R&DSystems) for 1 hour. The secondary antibodies used were anti-human AlexaFluor 488 dye conjugate (Life Technologies). Nuclei were stained with4′,6-diamidino-2-phenylindole (DAPI blue) (Life Technologies). Thefluorescence intensity of Alexa Fluor 488 dye was then measured using amicroplate reader Synergy Neo (BioTeK, Winooski, Vt., USA) andnormalized to the intensity by total protein quantity. To take an image,after mounting, the cells were visualized using a confocallaser-scanning microscope (Carl Zeiss).

Co-Culture Experiments and IL-2 Expression Measurement.

Co-culture of Jurkat T cells and tumor cells and IL-2 expressionmeasurement was performed as described previously (Sheppard, K. A. etal., 2004, FEBS letters, 574:37-41). To analyze the effect of tumorcells on T cell inactivation, tumor cells were co-cultured withactivated Jurkat T cells expressing human PD-1 which were activated withDynabeads® Human T-Activator CD3/CD28 (Life Technologies). Co-culturesat 5:1 (Jurkat:tumor cell) ratio were incubated for 12 or 24 hours.Secreted IL-2 level in medium was measured as described by themanufacturer (Human IL-2 ELISA Kits, Thermo Scientific).

Glycosylation Analysis of PD-L1.

To confirm glycosylation of PD-L1 protein, cell lysates were treatedwith the enzymes PNGase F, Endo H, O-glycosidase (New England BioLabs,Ipswich, Mass., USA) as described by the manufacturer. To stainglycosylated PD-L1 protein, purified PD-L1 protein was stained using theGlycoprotein Staining Kit (Peirce/Thermo Scientific) as described by themanufacturer.

Immunohistochemical Staining of Human Breast Tumor Tissue Samples.

Immunohistochemical (IHC) staining was performed as described previously(Lee et al., 2007, Cell, 130:440-455; Lo et al., 2007, Cancer Research,67:9066-9076; Chang, C. J. et al., 2011, Cancer cell, 19:86-100).Briefly, tissue specimens were incubated with antibodies against PD-L1,EGFR, B3GNT3, or Granzyme B and a biotin-conjugated secondary antibodyand then were incubated with an avidin-biotin-peroxidase complex.Visualization was performed using amino-ethylcarbazole chromogen. Forstatistical analysis, Fisher's exact test and Spearman rank correlationcoefficient were used; a p-value less than 0.05 was consideredstatistically significant. According to histological scoring, theintensity of staining was ranked into four groups: high (score 3),medium (score 2), low (score 1) and negative (score 0).

Identification of N-Glycopeptide.

Purified His tagged PD-L1 protein was reduced with 10 mM dithiothreitol(DTT) at 37° C. for 1 hour, alkylated with 50 mM iodoacetamide in 25 mMammonium bicarbonate buffer for 1 hour in the dark at room temperature,and then treated overnight with sequencing grade trypsin at anenzyme-to-substrate ratio of 1:50 at 37° C. The digested products werethen diluted with formic acid to a final concentration with 0.1%, andfurther cleaned up by ZipTip C18 (Millipore) before LC-MS/MS analysis.LC-MS/MS data were acquired at the Academia Sinica Mass SpectrometryFacility at IBC. The peptide mixture was analyzed by nanospray LC-MS/MSon an Orbitrap Fusion Tribrid (Thermo Scientific) coupled to an UltiMate3000 RSLCnano System (Dionex) with trap column Acclaim PepMap 100 (2cm×100 μm i.d) (Dionex). Peptide mixtures were loaded onto an AcclaimPepMap RSLC 25 cm×75 μm i.d. column (Dionex) and separated at a flowrate of 500 nL/min using a gradient of 5% to 35% solvent B (100%acetonitrile with 0.1% formic acid) for 60 minutes. Solvent A was 0.1%formic acid in water. The parameters used for MS and MS/MS dataacquisition under the HCD parallel with CID mode were: top speed modewith 3 s cycle time; FTMS: scan range (m/z)=400-2000; resolution=120 K;AGC target=2×10⁵; maximum injection time (ms)=50; FTMSn (HCD): isolationmode=quadrupole; isolation window=1.6; collision energy (%)=30 withstepped collision energy 5%; resolution=30 K; AGC target=5×10⁴; maximuminjection time (ms)=60; ITMSn (CID): isolation mode=quadrupole;isolation window=1.6; collision energy (%)=30; AGC target=1×10⁴. Rawdata were converted to Mascot generic format (MGF) by ProteomeDiscoverer 1.4. For glycopeptide identification, the HCD MS² data weresearched using Byonic (version 2.0-25) with the following searchparameters: peptide tolerance=2 ppm; fragment tolerance=6 ppm; missedcleavages=1; modifications: carbamidomethyl cysteine (fixed), methionineoxidation (common 2), deamidation at N (rare 1). The glycopeptide hitssuggested by Byonic were further checked manually by combining HCD andCID MS² results.

Statistical Analysis.

Data in bar graphs represents mean fold change relative to untreated orcontrol groups with standard deviation of three independent experiments.Statistical analyses were performed using SPSS (Ver. 20, SPSS, Chicago,Ill.). The correlation between protein expression and BLBC subset wasanalyzed using Spearman's correlation and Mann-Whitney test. Student's ttest was performed for experimental data. A P value <0.05 was consideredstatistically significant.

Example 2 PD-L1 Protein Expression Analysis

To elucidate the underlying mechanism of PD-L1, the protein expressionof PD-L1 was examined in human tumor tissues and cancer cell lines.FIGS. 1A and 1B and FIGS. 3A-3D illustrate protein expression in lung,breast, colon and ovarian cancer cell lines by Western blot analysis;FIG. 4A shows binding of PD-L1 protein in cells by different PD-L1antibodies. It was observed that a majority of PD-L1 protein wasdetected at ˜45 kDa (black circle), but a smaller fraction also appearedat 33 kDa (black arrowhead). Knocking down PD-L1 by lentiviralshort-hairpin RNA (shRNA) targeting either the coding sequence(shPD-L1#1) or the 3′UTR (shPD-L1#5) downregulated expression of boththe 33- and 45-kDa forms of PD-L1 (FIG. 4B). Reconstitution of PD-L1restored expression of both forms in the shPD-L1#5 clone (FIG. 1C; FIG.4C shows the vector design). These results showed that both bands on theWestern blot are PD-L1 protein and that the higher molecular weight formof PD-L1 is indicative of posttranslational modifications.

Glycosylated proteins frequently produce heterogeneous patterns in theWestern blot, as observed for the higher molecular weight (˜45 kDa) ofPD-L1. To test whether the glycosylation pattern observed for PD-L1corresponded to the glycosylated form, MDA-MB 231 and HeLa cells weretreated with recombinant glycosidase (Peptide-N-Glycosidase F; PNGase F)to remove N-glycan structure and then were subjected to Western blotanalysis. As shown in FIG. 4D, a significant portion of the 45-kDa PD-L1was reduced to the 33-kDa form of PD-L1 upon PNGase F treatment.Consistently, positive staining of the glycan structure was observed inpurified His-tagged PD-L1, but not in the presence of PNGase F (FIG.1D). These results demonstrate that the higher molecular weight of PD-L1is indeed the glycosylated form of the PD-L1 protein.

To recapitulate the expression of PD-L1 protein in cells, variousoverexpression constructs were generated to mimic endogenous expressionof the protein. To avoid possible cleavage at the N-terminal signalingpeptide, different tag sequences were fused at either the N- or theC-terminus (FIGS. 5A and 5B, above the blot). Similar to the resultsfrom endogenous PD-L1 expression analysis, transient transfection of allGFP-, HA-, Flag- or Myc-tagged PD-L1 had a ˜15 kDa molecular-weightshift from its actual size on the Western blot (FIGS. 1E and 5A and 5B).In contrast to PNGase F treatment, which removes the all of the N-glycanstructure on the PD-L1 protein, the addition of recombinant glycosidase,endoglycosidase H (Endo H), only partially reduced PD-L1 glycosylation,suggesting that complex types of N-linked glycan structures (containingboth high mannose and hybrid types) exist predominantly on PD-L1.Furthermore, glycosylation of PD-L1 was completely inhibited when cellswere treated with the N-linked glycosylation inhibitor, tunicamycin(TM), (FIGS. 1F and 5A-5D), but not 0-glycosidase (FIG. 5E). Together,these results indicate that PD-L1 is extensively N-linked glycosylatedin the cells tested (Heifetz, A., et al., 1979, Biochemistry,18:2186-2192).

Example 3 Glycosylation Analysis

Western blot analysis using two PD-L1-specific antibodies (anti-PD-L1and anti-hB7-H1) indicated that PD-L1 glycosylation occurred on itsextracellular domain (ECD, recognized by anti-hB7-H1) but not on itsintracellular domain (ICD, recognized by anti-PD-L1) (FIGS. 1F and 5C).To pinpoint the glycosylation sites, a sequence alignment of the PD-L1amino acid sequences from different species was performed to search forevolutionarily conserved NXT motifs, a consensus N-glycosylationrecognition sequence (Schwarz, F., et al., 2011, Current opinion instructural biology, 21:576-582). Consistent with the earlier prediction(Cheng et al., 2013, JBC, 288:11771-85); and Vigdorovich et al., 2013,Structure, 21:707-17), four NXT motifs were identified (FIG. 1G andFIGS. 6A and 6B). To confirm if these sequences were indeedglycosylated, the tryptic peptides of a purified human PD-L1 wereanalyzed by nano LC-MS/MS. Glycopeptides carrying complex type N-glycanswere identified for each of the 4 N-glycosylation sites (FIGS. 7A-71I),consistent with the apparent resistance to Endo H treatment (FIG. 1E). Aseries of asparagine (N) to glutamine (Q) substitutions were generatedto determine the specific glycosylation site(s) on the PD-L1 protein.All four mutants, N35Q, N192Q, N200Q, and N219Q, exhibited a certaindegree of reduction in glycosylation compared with the WT PD-L1 (FIG.1H, lanes 2, 3, 4, and 5). No detectable differences in glycosylationwere observed for the three non-NXT NQ PD-L1 mutants (FIG. 1H, lanes 11,12, and 13). In addition, PD-L1 glycosylation was completely ablated inthe PD-L1 4NQ variants in which all four asparagines were mutated toglutamine as indicated by the absence of signals corresponding to theglycosylated form at 45 kDa (FIG. 1H, lane 10 (4NQ) and lane 14 (WT)).Based on the crystal structure of the PD-1/PD-L1 complex (Lin, D. Y., etal., 2008, Proceedings of the National Academy of Sciences of the UnitedStates of America, 105:3011-3016, these four glycosylation sites ofPD-L1 (N35, N192, N200 and N219) are exposed on the surface of theprotein. Mutation of the PD-L1 glycosylation sites (PD-L1 4NQ) did notaffect the overall structure based on the prediction. These resultssuggest that PD-L1 exists exclusively as an N-glycosylated glycoproteinin the cells and that all four NXT motifs are glycosylated.

The levels of glycosylated PD-L1 observed were significantly higher thanthe non-glycosylated form of the PD-L1 protein (FIGS. 1A and 1B and 5Cand 5D). Based on this finding, the inventors investigated whetherglycosylation stabilizes the PD-L1 protein. To this end, the proteinturnover rate was measured in the presence of protein synthesisinhibitor cycloheximide (CHX) to determine the effect of glycosylationon PD-L1 protein stability. The turnover rate of Flag-tagged PD-L1 wasmuch faster in tunicamycin (TM)-treated than in DMSO-treated HEK 293Tcells (FIGS. 2A; 8A and 8B). Similar results were observed for theendogenous PD-L1 in A431 cells (FIG. 8C). Under proteasome inhibition byMG132, the levels of non-glycosylated PD-L1 steadily increased over a12-h period (FIG. 2B). Consistently, non-glycosylated PD-L1 4NQ wasrapidly degraded with a half-life as short as 4 hours (FIG. 2C), similarto that of the non-glycosylated PD-L1 WT (FIG. 2A), suggesting thatimpaired glycosylation leads to degradation of PD-L1 via the 26Sproteasome.

Example 4 Binding Affinity of PD-L1

PD-L1 is a key immune suppressor through its binding with PD-1 duringcancer progression (Okazaki, T., et al., 2013, Nature immunology,14:1212-1218. Thus, the binding affinities of WT PD-L1 andglycosylation-deficient mutant PD-L1 to PD-1 were compared. PD-L1 WT andthe 4NQ mutant PD-L1 were stably expressed in MDA-MB-468 and HEK-293Tcells, and stable clones with similar amounts of PD-L1 WT and 4NQexpression were selected and then incubated with recombinant PD-1/Fcfusion protein, followed by the addition of anti-human IgG (Fc specific)fluorescence conjugate for signal amplification (Cheng, X. et al., 2013,The Journal of biological chemistry, 288:11771-11785) (FIG. 8D). Whilethere were no significant changes in membrane localization betweenglycosylated and non-glycosylated PD-L1 (FIGS. 8E (confocal image) and8F (biotinylation pull-down)), a marked difference was observed in PD-1binding on the cell membrane between stable transfectants treated withor without TM (FIG. 2D, quantification graph shown on the right).Furthermore, ablation of PD-L1 glycosylation by TM treatment, orexpression of the 4NQ mutant, reduced the association of PD-L1 with PD-1(FIGS. 2E and 8G). In vitro binding experiments also demonstrated theloss of PD-L1 4NQ/PD-1/Fc interaction, suggesting that glycosylation ofPD-L1 enhances its association with PD-1 (FIG. 2F). Becauseimmunosuppression activity is associated with cell-cell interactioninvolving cytotoxic T-cells, a co-culture experiment was performed tomeasure the IL-2 expression levels, an indicator of T-cell immuneresponse that is suppressed by the PD-L1 and PD-1 interaction, in JurkatT-cells after exposure to PD-L1 stable clones (Yang, W., et al., 2008,Investigative ophthalmology & visual science, 49:2518-2525) (FIG. 8H).Consistently, the loss of PD-L1 glycosylation impaired its interactionwith PD-1 and enhanced the release of soluble IL-2 (FIG. 2G). Theseresults reveal that the integrity of the PD-L1 glycophenotype isrequired for its immunosuppressive function.

Example 5 Functionality of the PD-L1 Glycophenotype

PD-L1 WT and 4NQ mutants were stably expressed in endogenousPD-L1-depleted MDA-MB-468 and BT549 cells. Using these cell lines, PD-1and PD-L1 binding affinity were analyzed. As shown, the associationbetween glycosylation variant PD-L1 4NQ and PD-1 was decreased (FIG.9A). In vitro binding experiments further demonstrated thatglycosylation is required for the PD-L1 and PD-1 association (FIG. 9B).T cell-mediated tumor cell killing was measured in vitro by co-culturingBT549 PD-L1 WT and PD-L1 4NQ expressing stable cell lines with humanprimary T cells (time-lapse microscopy). Consistent with the loss ofPD-1 binding, the PD-L1 4NQ stable cell line showed more T cell-mediatedkilling of cancer cells (FIG. 9C). In addition, the immunosuppressivefunction of PD-L1 was measured in vivo in a syngeneic 4T1 mouse model inwhich tumor growth was measured in BALB/c mice that had received eitherPD-L1 WT or PD-L1 4NQ expressing 4T1 cells. Compared with mice havingcells that expressed PD-L1 WT, the mice having cells that expressedPD-L1 4NQ showed reduced tumor size and more activated cytotoxic T cells(FIGS. 9D and 9E). Together, these data demonstrate that the loss ofPD-L1 glycosylation impairs its interaction with PD-1 and impairs theability of the PD-1/PD-L1 interaction to allow tumor cells to evadeimmune surveillance by T-cells. Accordingly, tumor cells in which PD-L1is non-glycosylated, or is aberrantly glycosylated, provide targets thatare amenable to be killed by functional effector T-cells. This mechanismof preventing or blocking tumor cells from escaping T-cell immunesurveillance by impaired glycosylation of their membrane-expressed PD-L1further corroborates that the integrity of the PD-L1 glycophenotype isrequired for its immunosuppressive function.

In addition, the syngeneic 4T1 BALB/c mouse model as above was used todetermine the effect of a representative dual function antibody, namely,STM073, on tumor growth (i.e., the immunosuppressive function) in theanimals during a 16-day study period. Briefly, in the study, animalsreceived 4T1 cells expressing wild type (glycosylated) PD-L1 (1×10⁵ 4 T1cells) on Day 0. On Days 3, 5, 7, 9, 11 and 13 thereafter, the4T1-animals were treated with STM073 MAb (100 μg) or IgG controlantibody (100 μg) by injection. During the treatment period, mortalityand tumor volume were assessed in the animals. FIG. 9F presents boxgraphs showing that tumor volume in the mice that were treated with theSTM073 (“73”) MAb was measurably lower compared with the tumor volume incontrol animals.

Example 6 Production of Glycosylated PD-L1-Binding Monoclonal Antibodies

Hybridomas producing monoclonal antibodies generated againstglycosylated human PD-L1 were obtained by the fusion of SP2/0 murinemyeloma cells with spleen cells isolated from human PD-L1-immunizedBALB/c mice (n=6) (Antibody Solution, Inc.) according to standardizedprotocol. Before fusion, sera from the immunized mice were validated forbinding to the PD-L1 immunogen using FACS analysis. Monoclonal antibody(MAb)-producing hybridomas were generated and the isotype of all of theMAbs was IgG1. The hybridomas that produced antibodies were again testedfor specificity.

To identify anti-glycPD-L1 MAbs that were specific for and whichpreferentially bound glycosylated PD-L1 antigen (i.e., glycosylatedPD-L1 specific MAbs) versus non-glycosylated PD-L1, different types ofassays were performed. In a screening assay to detect preferentialbinding of MAbs to glycosylated PD-L1, antibody binding was determinedbased on the measurement of fluorescence intensity through FACS analysis(using cell membrane bound proteins). By way of example, the assay wasperformed using the BT549 human breast cancer cell line. Illustratively,BT549 cells overexpressing PD-L1 WT (fully glycosylated) were labeledwith biotin according to conventional procedures and then mixed withBT549 cells overexpressing PD-L1 4NQ (fully unglycosylated PD-L1variant). The mixed cells were incubated with anti-PD-L1 antibodies,e.g., anti-glycPD-L1 antibodies, and were further incubated withsecondary antibodies conjugated with FITC as detection agent. Afterwashing, fluorescence intensity (measured fluorescence intensity, MFI)was measured via FACS/flow cytometry analysis to assess the relativebinding of the anti-PD-L1 antibodies to membrane bound PD-L1 WT (oncells) or to 4NQ PD-L1 (on cells). Antibodies that exhibitedsignificantly higher MFI on WT PD-L1 versus 4NQ PD-L1 were selected forfurther evaluation. Results for the fluorescence binding analysis of theSTM073 and STM108 dual function MAbs are presented in Table 6 below,which shows the MFI values for antibody binding to BT549 cellsexpressing wild type (glycosylated) PD-L1, (BT549PD-L1WT Cells) versusantibody binding to BT549 cells expressing variant (non-glycosylated4NQ) PD-L1, (BT549PD-L1 4NQ Cells). The experimental results in Table 6show an approximately 5-fold higher MFI value for STM073 MAb binding toBT549PD-L1WT cells (glycosylated PD-L1-expressing cells) compared withBT549PD-L1 4NQ cells (non-glycosylated PD-L1-expressing cells).Similarly, an approximately 4-fold higher MFI value was determined forSTM108 binding to BT549PD-L1WT cells compared with BT549PD-L1 4NQ cells.

TABLE 6 Measured Fluorescence Intensity Values for Anti-glycPD-L1 MAbsMFI MFI MAb (BT549PD-L1WT Cells) (BT549PD-L1 4NQ Cells) STM073 63.9012.21 STM108 117.42 27.57Based on the binding analysis, forty-two candidate MAb-producinghybridomas were selected, grown in ADCF medium, and their supernatantcontaining monoclonal antibody was concentrated and purified.

In some cases, the purified MAbs were further tested for their abilityto neutralize or inhibit the interaction between PD-L1 and PD-1(PD-L1/PD-1 interaction) using a live-cell imaging assay, IncuCyte™,(Essen Bioscience). For this assay, BT-549 cells expressing PD-L1 wereincubated with anti-human PD-L1 antibody and with fluorescent-labeledPD-1-Fc fusion proteins. Ligand and receptor binding was quantified byIncuCyte™ Zoom every hour, according to the manufacturer's instructions.Based on this assay, it was found that of the 42 MAbs tested, 15 MAbscompletely blocked the binding of PD-L1 to PD-1. Some of the 15 MAbsthat showed strong blocking efficacy also bound non-glycosylated PD-L1to some extent.

In another assay, both glycosylated human PD-L1 protein andnon-glycosylated PD-L1, i.e., PD-L1 protein treated with PNGase F, werecoated onto a solid phase and tested for binding affinity of the MAbs tothe PD-L1 antigens. It will be understood that “PD-L1 antigen” issynonymous with “PD-L1 protein.” Twelve (12) of the MAbs showed a higheraffinity interaction with glycosylated PD-L1 protein compared tonon-glycosylated PD-L1 protein (PNGase F treated protein). For furtherspecificity analysis, selected MAbs were analyzed by Western Blot andFACS flow cytometry analysis. From the various analyses, MAbs, such asSTM073 and STM108, were found to specifically bind the glycosylated formof PD-L1 compared with the non-glycosylated form of PD-L1, which furthervalidated the specificity of these MAbs for glycosylated PD-L1 antigen.

Example 7 Identification of Binding Regions of Specific GlycosylatedPD-L1-Binding Monoclonal Antibodies

To identify the regions of monoclonal anti-glycPD-L1 antibodies whichbound to glycosylated PD-L1, wild type (glycosylated) PD-L1 (PD-L1 WT),and the glycosylation variant proteins N35/3NQ, N192/3NQ, N200/3NQ, andN219/3NQ (in which one of the glycosylation sites at position 35, 192,200 or 219 is the wild type asparagine (N) at that position and theother three positions have been mutated to glutamine (Q) have beenmutated so it is not glycosylated) (FIG. 10A) were overexpressed inPD-L1 knockdown BT549 cells. As determined by Western blot, some MAbsrecognized particular PD-L1 mutants with higher levels of bindingcompared with other PD-L1 mutants, demonstrating that such MAbs weresite-specific. For example, MAb STM073 bound the mutants N35/3NQ,N192/3NQ and N200/3NQ, but not mutant N219/3Q, demonstrating that thisantibody bound to the N35, N192 and N200 regions of PD-L1, but not toN219 (FIG. 10B). Further, Western blot analysis using liver cancer celllysate also revealed a differential pattern of PD-L1 glycosylation for arepresentative anti-glycPD-L1 antibody such as STM073 (FIG. 10C).

The histopathologic relevance of these MAbs was further demonstrated byimmunohistochemical (IHC) staining. In a cytospin staining analysis, theanti-glycPD-L1 monoclonal antibodies consistently recognized and boundthe glycosylated portion of the PD-L1 protein, but not unglycosylatedPD-L1 protein. In a human triple negative breast cancer patient sample,the anti-glycPD-L1 monoclonal antibodies also showed membrane andcytoplasm staining in a 1:30 ratio. These data demonstrated that theanti-glycPD-L1 monoclonal antibodies can be used in biomarker analysesfor detection of glycosylated PD-L1 as biomarker.

Example 8 Epitope Mapping of Glycosylated PD-L1-Binding Antibodies

Epitope mapping for the mouse monoclonal anti-glycPD-L1 antibody STM073was performed by CovalX AG (Switzerland). To determine the nature of theepitope, e.g., either linear or conformational, recognized by theanti-glycPD PD-L1 antibodies generally, and the STM073 MAb inparticular, studies were conducted to evaluate whether the interactionbetween the PD-L1 antigen proteins and the anti-PD-L1 antibodies couldbe inhibited by unstructured peptides generated by proteolysis of theantigen. If the peptides generated by complete proteolysis of the PD-L1antigen are able to inhibit the binding of the antigen by the antibody,the interaction is not based on conformation, and the epitope is linear.A simple competition assay with a bank of overlapping peptides generatedfrom the sequence of the antigen is sufficient to determine the sequenceof the epitope. Alternatively, if the peptides generated by completeproteolysis of the PD-L1 antigen are unable to inhibit the binding ofthe antigen by the antibody, the conformation of the target isdetermined to be necessary for interaction, and the epitope isconformational, e.g., continuous (with a special conformation such as aloop) or discontinuous (due to tertiary structure). To further elucidatebinding to a conformational epitope, covalent labeling, peptide mapping,and high resolution mass spectrometry were also employed.

Competition assays showed that peptides generated from the PD-L1 antigendid not inhibit the binding of MAb STM073 to the PD-L1 antigen,confirming that the STM073 MAb epitope is conformational and not linear.Using chemical cross-linking, High-Mass MALDI mass spectrometry andnLC-Orbitrap mass spectrometry, the interaction surfaces between thePD-L1 protein and the antibodies were characterized. Competition assaysusing pepsin proteolysis of PD-L1, mixture of the resultingpepsin-generated PD-L1 peptides with antibodies and intact PD-L1protein, and analysis of the antigen/antibody interaction by knownmethods showed no detectable inhibition of binding of the STM073monoclonal antibody to the PD-L1 antigen by the PD-L1 peptides.Accordingly, the epitope on PD-L1 recognized by the anti-PD-L1 MAbSTM073 was determined to be conformational and not linear. The epitopeof STM073 was determined to include amino acid residues H69, Y112, R113and K124, which are underlined in the sequence starting with V68 andending with V128 (with the region within positions 79 to 107 indicted asdashes) VHGEEDLKVQH------DAGVYRCMISYGGADYKRITV (SEQ ID NO: 85). STM108was found to bind an epitope within the sequenceLKVQHSSYRQR------EGYPKAEVIWTSSDHQ, which are amino acids 74 to 84 and158 to 173, respectively, of SEQ ID NO:1, and contacts residues S80,S81, K162 and S169 of SEQ ID NO. 1, within residues 74 to 173 of SEQ IDNO: 1.

Example 9 T Cell Killing Assay

T cell killing assays were utilized to determine the cytotoxic activityof anti-glycPD-L1 monoclonal antibodies as described herein on tumorcells. The protocol followed is as follows: On Day 0, serum-containingmedium was removed from glycosylated wild type PD-L1- (PD-L1 WT)expressing BT549 RFP target cell cultures and gently rinsed twice withPBS. Cells were harvested and counted. The cell suspension wascentrifuged (1000 RPM, 4 minutes) and the cell pellet was resuspended inculture medium at 50,000 cells/mL. Using a manual multichannel pipette,the cells were seeded (100 μL/well, i.e., 5000 cells/well) into everywell of a flat-bottom microplate. The plate was allowed to stand atambient temperature for 30 minutes and then was positioned into anIncuCyte ZOOM® live-cell imager where it was left to equilibrate for 20minutes before scheduling the first scan. Twenty-four hour (24 hr)repeat scanning (10× objective) was scheduled for every 3 hours, withthe first scan commencing immediately. Cell confluence was monitored forthe next 18 hours (overnight) until the desired confluence (e.g., 20%)was achieved.

The next morning, the day of the assay (i.e., Day 1), a 10 μM solutionof IncuCyte™ Caspase 3/7 apoptosis green fluorescence detection reagent(Essen BioScience 4440) was prepared in assay medium (4× final assayconcentration of 2.5 μM) and warmed to 37° C. in an incubator. Ananti-CD3 antibody (100 ng/mL)+IL-2 (10 ng/mL) T cell activator treatmentwas prepared at 4× final assay concentration in assay medium and warmedto 37° C. Test MAbs were also prepared. The target cell plate wasremoved from the incubator and the medium was aspirated, taking care notto damage the cell layer. Using a multichannel pipette, 25 μL of thewarmed caspase 3/7 solution was transferred into each well. Thereafter,25 μL of the warmed anti-CD3 antibody+IL-2, and the antibodies, wereplaced into the appropriate wells of the cell plate. An additional 50 μLof medium containing the effector cells (PBMCs or Total T cells) wasadded to form a total assay volume of 100 μL. The de-bubbled cell platewas positioned in the IncuCyte ZOOM® instrument and allowed toequilibrate for 20 minutes prior to the first scan. 24-hr repeatscanning was scheduled for every 2-3 hours for up to 5 days. (Objective10×; Vessel Type: Corning 3596; Scan Mode: Standard; Scan Pattern: 2images per well; Channel: Phase+“Green” (+“Red” if NucLight™ Red targetcells were used)).

For analysis, target-cell apoptosis was quantified in the IncuCyte™software by counting the total number of “large” green-fluorescentobjects (nuclei) in the field of view over time. The proliferation oftarget cells was measured from the red object count, corresponding tothe number of red cell nuclei. Data were expressed as the number offluorescent objects per mm². Data showed that addition of antibodies asdescribed herein, specifically STM073, enhanced tumor cell killing (FIG.11).

Example 10 Binding Assay

To determine whether an anti-glycPD-L1 monoclonal antibody as describedherein specifically inhibits the interaction of PD-1 and PD-L1, thefollowing binding assay was performed. On Day 0 of the assay,serum-containing medium was removed from PD-L1-expressing BT549 targetcell culture and gently rinsed twice with D-PBS. Cells were harvestedand counted. The cell suspension was centrifuged (1000 RPM, 5 minutes)and the cell pellet was resuspended in culture medium at 50,000cells/mL. A manual multichannel pipette was used to seed the cells (100μL/well, i.e., 5000 cells/well) into every well of a flat-bottommicroplate. The plate was allowed to stand at ambient temperature for 30minutes. Thereafter, the plates containing the cells were incubatedovernight in a 5% CO₂ incubator.

On Day 1 of the assay (i.e., the next morning), culture mediumcontaining 1 μg/mL PD-1/Fc and a 1:400 dilution of Alex Fluor 488-goatanti-human IgG was prepared and warmed to 37° C. in an incubator. Thecell plate was removed from the incubator and the medium was aspirated,taking care not to damage the cell layer. 50 μL of test antibody wasadded to each well in a dose-dependent manner. 50 μL of the culturemedium containing PD-1/Fc and Alex Fluor 488-goat anti-human IgG wasadded to every well. The cell plate was positioned in the IncuCyte ZOOM®instrument and allowed to equilibrate for 20 minutes prior to the firstscan. 24-hr automated repeat scanning (10×) was scheduled for every 1-2hours for up to 24 hours. Objective: 10×; Vessel Type: Corning 3596;Scan Mode: Standard; Scan Pattern: 4 images per well; Channel:Phase+“Green”.

FIGS. 12A and 12B show that representative MAbs STM073 and STM108inhibited binding of PD-1/Fc to cells expressing PD-L1 in a dosedependent manner. The control assay results are shown in FIG. 12C.

Example 11 PD-L1 Internalization Assay

To determine whether an anti-glycPD-L1 monoclonal antibody promotesPD-L1 internalization and degradation, A431 cells were incubated inserum free medium overnight and then incubated with 10 μg ofanti-glycPD-L1 antibody for two days. The cells were then harvested, andPD-L1 in the cells was assessed by Western blot. FIG. 13 shows thatincubation with the STM073 shows a reduced level of PD-L1 in the cellscompared to control (IgG).

The cellular internalization of the STM073 and STM108 MAbs wasvisualized by labelling the antibodies with pHrodo™ Red dye using thepHrodo™ Red Microscale Labeling Kit (ThermoFischer Scientific,Rochester, N.Y.) according to the manufacturer's instructions. Briefly,for the analysis, cells were seeded at time 0; at 24 hours afterseeding, cells were incubated with labeled STM073 or STM108 MAbs (5μg/mL). After 1 hour, an image scan of the cells was begun using anIncuCyte ZOOM® instrument with scheduled 24-hour repeat scanning (10×)for every 1 hour. Objective: 10×; Vessel Type: Corning 356407; ScanMode: Standard; Scan Pattern; 3 images per well; Channel: Phase+“Red.”FIG. 14A: Wild type BT549 cells (human ductal carcinoma, breast cancercell line) incubated with STM073; FIG. 14B: BT 549 cells overexpressingPD-L1 WT (glycosylated) incubated with STM073; FIG. 14C: NCI-H226 cells(human lung cancer cell line, squamous cell mesothelioma) incubated withSTM073; FIG. 14D: MCF-7 cells (human breast cancer cell line,adenocarcinoma) incubated with STM073; and FIG. 14E: BT 549 cellsexpressing PD-L1 WT (glycosylated) incubated with STM108.

Example 12 Binding of PD-L1 by Anti-glycPD-L1 Antibodies Promotes PD-L1Internalization and Degradation

FIGS. 15A-15C provide an example of PD-L1 internalization anddegradation via live cell imaging analysis following binding ofanti-glycPD-L1 MAb STM108 to PD-L1 expressed on BT549-PD-L1 cells. InFIGS. 15A-15C, the anti-PD-L1 antibody is STM108 conjugated to a redfluorescent dye, pHrodo™ Red (succinimidyl ester (pHrodo™ Red, SE) usingthe pHrodo™ Red Microscale Labeling Kit, (ThermoFisher Scientific,Rochester, N.Y.), as described above in Example 11. Green stainingreflects cells stained with LysoTracker® Green DND-26, which is a cellpermeable green dye that stains acidic compartments (lysosomes) in livecells imaged via live cell imaging. FIG. 15A shows that at a first timepoint (Time 0), the STM108 antibody is internalized into cells asobserved by the intense red intracellular staining of cells indicated bythe arrow. FIG. 15B shows the weakened intracellular red staining in thesame cells depicted in FIG. 15A, at a time 2 minutes after Time 0 inFIG. 15A. FIG. 15C shows the lack of red intracellular staining 4minutes after Time 0 in FIG. 15A, which reflects the degradation of theSTM108 antibody and/or the antibody-antigen complex inside the cells.These images reflect that an anti-glycPD-L1 antibody such as STM108 MAbeffectuates internalization and degradation of PD-L1 after binding toPD-L1 expressed on the cell surface.

Example 13 Internalization of PD-L1 Bound by Anti-glycPD-L1 Antibodiesin Tumor Cells Versus Total T Cells

Anti-glycPD-L1 antibodies were tested for the ability to internalizeinto PD-L1 positive tumor cells after binding cell-surface expressedPD-L1, as compared to activated or non-activated T cells. Theanti-glycPD-L1 antibodies STM004, STM073 and STM108, and mouse IgG ascontrol were incubated with non-activated total T cells from peripheralblood, activated total T cells from peripheral blood and NCI-H226 cells,which express PD-L1. For T cell activation, total T cells were mixedwith beads, e.g., inert, superparamagnetic beads, covalently coupledwith anti-CD3 and anti-CD28 antibodies (e.g., ThermoFisher Scientific,Rochester, N.Y.) at a 1:1 ratio to stimulate T cells in a manner mimicsstimulation by antigen-presenting cells (See, e.g., A. Trickett et al.,2003, J. Immunol. Methods, 275, Issues 1-2:251-255). All antibodies werelabeled with pHrodo™ Red and internalization was visualized as describedin Example 11. FIGS. 16A-16D and FIGS. 16E-16H show that none of theantibodies tested were internalized into non-activated total T cells oractivated total T cells. FIGS. 16I-16L show that the dual function,internalizing STM073 and STM108 MAbs were internalized into NCI-H226cells following incubation with these cells, as evidenced by redintracellular staining, compared with the labeled control antibody, mIgG(FIG. 16I) and with labeled non-internalizing STM004 MAb. (FIG. 16J),which showed no red intracellular staining. This example demonstratesthat the dual function anti-glycPD-L1 antibodies are selectivelyinternalized into PD-L1-expressing tumor cells but are not internalizedinto either activated or non-activated T cells.

Example 14 Efficacy of PD-L1 ADC in Tumor Cell Killing and Reduction ofTumor Volume

Both in vitro and in vivo experiments were conducted to evaluate theefficacy of an ADC comprising an anti-human glycPD-L1 MAb, i.e., STM108MAb, coupled to the cytotoxin MMAE in tumor killing and in reducingtumor volume. The STM108-ADC comprises the STM108 MAb chemically linkedvia cysteines to MC-vc-PAB-MMAE, as described hereinabove, for specificdelivery of the MMAE cytotoxin payload to PD-L1-expressing tumor orcancer cells. Measured physical properties of the STM108-ADC(STM108-MC-vc-PAB-MMAE) are as follows:

A_(248nm) of ADC 2.548 A_(280nm) of ADC 3.635 Ratio A_(248nm)/A_(280nm)0.70 Drug Antibody Ratio 4.13

In connection with this Example, FIGS. 17A-17D present the results ofexperiments conducted to evaluate the efficacy of STM108-ADC in killingPD-L1-expressing and non-PD-L1-expressing tumor cells and in reducingthe volume of tumors in tumor-grafted mice as compared to controls (IgGand STM108 MAb alone). FIG. 17A shows the % viability ofPD-L1-expressing MDA-MB231 (human breast carcinoma cell line) tumorcells (“MB231”) following exposure to different concentrations (nM) ofSTM108-ADC (filled black circles) compared with the % viability of MB231cells molecularly engineered to knock out their expression of PD-L1(“MB231 PDL1 KO”) following exposure to different concentrations ofSTM108-ADC, i.e., “ADC108” (filled black squares). As observed, theviability of MB231 cells that did not express PD-L1 on their surface wasnot significantly affected by STM108-ADC at even the highestconcentration, while the viability of PD-L1-expressing MB231 cells wassignificantly reduced, particularly at STM108-ADC concentrations of 1 nMup to 100 nM. In FIG. 17B, an MDA-MB231 mouse model of breast cancer wasused in which animals grafted with tumors derived from MB231 cells weretreated with either an IgG-MMAE control (100 μg); with STM108-ADC at 50at 100 μg, or at 150 μg; or with STM108 MAb alone (100 m). The resultsdemonstrated that tumor volume was effectively decreased intumor-bearing animals treated with STM108-ADC at all dose levels, aswell as, to some extent, with STM108 MAb (100 μg), compared with animalstreated with the IgG-MMAE control. In addition, it was surprisinglyfound that complete remission (“CR”) occurred in 3 out of 5 (3/5)animals treated with STM108-ADC (100 μg) and in 4 out of 5 (4/5) animalstreated with STM108-ADC (150 μg) by about day 18.

FIG. 17C shows the % viability of 4T1 mammary carcinoma cellsmolecularly engineered to express human PD-L1 on the cell surface (“4T1hPDL1”) following exposure to different concentrations (nM) ofSTM108-ADC (open red circles) compared with the % viability of 4T1 cellsthat naturally do not express PD-L1 (“4T1”) following exposure todifferent concentrations of STM108-ADC, i.e., “ADC108” (open redsquares). As observed, the viability of 4T1 cells that do not expressPD-L1 on their surface was not significantly affected by STM108-ADC ateven the highest concentration, while the viability of PD-L1-expressing4T1 cells was reduced at STM108-ADC concentrations of greater than 10 nMto 100 nM.

4T1 syngeneic mouse models of breast cancer were used in which theanimals (Balb/c mice) were grafted with tumors derived from 4T1 mammarycarcinoma cells that had been molecularly engineered to express PD-L1 orthe cell surface (“4T1 hPD-L1”), or in which the Balb/c mice weregrafted with tumors derived from untransfected 4T1 mammary carcinomacells that do not naturally express PD-L1 on the cell surface (“4T1”).All procedures with BALB/c mice (6- to 8-week-old females; JacksonLaboratories, Bar Harbor, Me., USA) were conducted under guidelinesapproved by the Institutional Animal Care and Use Committee at MDAnderson. Mice were divided according to the mean tumor volume in eachgroup. 4T1 cells (1×10⁵ cells in 25 μL of medium mixed with 25 μL ofMatrixgel Basement Membrane Matrix [BD Biosciences, San Jose, Calif.,USA]) were injected into the mammary fat pad. IgG-MMAE control (100 μg),STM108 MAb (100 μg), or STM108-ADC (150 μg) were injectedintraperitoneally on days 3, 5, 7, 9, 11, and 13 after tumor cellinoculation of mice. Tumors were measured every 3 days with a caliper,and tumor volume was calculated using the following formula:π/6×length×width.

As observed in FIG. 17D, in animals harboring 4T1-derived tumors havinglittle or no cell-surface PD-L1 expression (open circles/dotted lines),the results showed that tumor volume increased over time for alltreatment types, i.e., IgG-MMAE control, STM108 MAb, or STM108-ADC. Inanimals harboring tumors derived from PD-L1-expressing 4T1 cells andtreated with the IgG-MMAE control, tumor volume in treated animals alsoincreased over time (solid black circles). By contrast, in animalsharboring tumors derived from PD-L1-expressing 4T1 cells and treatedwith either the STM108 MAb (solid blue circles) or with STM108-ADC(solid red circles), tumor volume was effectively decreased. Inaddition, it was surprisingly found that complete remission (“CR”)occurred in 5 out of 7 (5/7) animals treated with STM108-ADC by aboutday 21.

The beneficial and effective antineoplastic and therapeutic aspectsrelated to the use of an ADC comprising an anti-glycPD-L1 MAb asdescribed hereinabove, such as STM108, in treating cancers, e.g., twotypes of breast tumors, are underscored by the in vivo results showingsignificant reduction in tumor volume and complete remission of tumorsin animals that had been treated with the anti-glycPD-L1 MAb ADC within25 days after treatment, e.g., by about 15-23 days.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods have been described in terms of embodimentsand preferred embodiments, it will be apparent to those of skill in theart that variations may be applied to the methods and in the steps or inthe sequence of steps of the methods described herein without departingfrom the concept, spirit and scope of that which is described. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of thedescribed embodiments as defined by the appended claims.

All patents, published patent applications, and other publications citedherein are hereby incorporated by reference in the present application.

What is claimed is:
 1. An isolated antibody which selectively binds toglycosylated PD-L1 relative to unglycosylated PD-L1, inhibits binding ofglycosylated PD-L1 to PD-1, and promotes internalization and degradationof PD-L1 on tumor cells.
 2. The isolated antibody of claim 1, which isrecombinantly engineered, chimeric, or humanized.
 3. The isolatedantibody of claim 1 or claim 2, which binds to glycosylated PD-L1 with aK_(d) that is less than half of the K_(d) exhibited by the antibodybinding to unglycosylated PD-L1.
 4. The isolated antibody of claim 3,which binds to glycosylated PD-L1 with a K_(d) at least 10 times lessthan the K_(d) exhibited by the antibody binding to unglycosylatedPD-L1.
 5. The isolated antibody of claim 4, which binds glycosylatedPD-L1 with an affinity of from 5-20 nM.
 6. The isolated antibody of anyone of claims 1 to 3, wherein the antibody, which is directly orindirectly detectable by a fluorescent label, preferentially binds tocells expressing glycosylated PD-L1 with a measured fluorescenceintensity (MFI) that is 2-fold to 10-fold higher than the MFI exhibitedby the antibody binding to cells expressing unglycosylated PD-L1 in acell flow cytometry assay.
 7. The isolated antibody of claim 6, whereinthe antibody preferentially binds to cells expressing glycosylated PD-L1with a MFI that is 3-fold to 5-fold or more higher than the MFIexhibited by the antibody binding to cells expressing unglycosylatedPD-L1.
 8. The isolated antibody of any one of claims 1 to 7, whichspecifically binds to an epitope of glycosylated PD-L1 comprising atleast one of amino acids H69, S80, Y81, Y112, R113, K162, K124 and S169of SEQ ID NO:
 1. 9. The isolated antibody of any of claim 8, wherein theepitope comprises amino acids within region V68 to V128 or within regionL74 to Q173 of SEQ ID NO:
 1. 10. The isolated antibody of any one ofclaims 1 to 7, which specifically binds to an epitope of glycosylatedPD-L1 comprising at least one of amino acids Y112, R113 and S117 of SEQID NO:
 1. 11. The isolated antibody of claim 10, wherein the epitopecomprises amino acids within region D108 to V128 of SEQ ID NO:
 1. 12.The isolated antibody of any one of claims 1 to 7, which specificallybinds to an epitope of PD-L1 recognized by the monoclonal antibody (MAb)STM073 or the MAb STM108.
 13. The isolated antibody of any one of claims1 to 7, which competes or cross competes for specific binding toglycosylated PD-L1 with MAb STM073 or MAb STM108.
 14. The isolatedantibody of any one of claims 1 to 7, which comprises the heavy chainvariable (V_(H)) domain or the light chain variable (V_(L)) domain ofMAb STM073 or MAb STM108.
 15. The isolated antibody of any one of claims1 to 7, which comprises heavy chain CDRs1-3 and/or light chain CDRs1-3from MAb STM073 or MAb STM108.
 16. The isolated antibody of claim 15,which has human antibody framework regions.
 17. The isolated antibody ofany one of claims 1 to 7, wherein the heavy chain variable domain(V_(H)) has an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:
 19. 18.The isolated antibody of any one of claim 1 to 7, or 17, wherein theV_(L) domain has an amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:27.
 19. The isolated antibody of any one of claims 1 to 7, wherein theV_(H) domain has an amino acid sequence of SEQ ID NO: 3 and the V_(L)has an amino acid sequence of SEQ ID NO:
 11. 20. The isolated antibodyof any one of claims 1 to 7, wherein the V_(H) domain has an amino acidsequence of SEQ ID NO: 19 and the V_(L) domain has an amino acidsequence of SEQ ID NO:
 27. 21. The isolated antibody of any one ofclaims 17 to 20, which comprises a human constant domain.
 22. Theisolated antibody of any one of claims 1 to 7, which has a V_(H) domaincomprising a CDR H1 with an amino acid sequence of SEQ ID NO: 4, a CDRH2 with an amino acid sequence of SEQ ID NO: 6, and a CDR H3 with anamino acid sequence of SEQ ID NO: 8, or a V_(H) domain comprising a CDRH1 with an amino acid sequence of SEQ ID NO: 5, a CDR H2 with an aminoacid sequence of SEQ ID NO: 7, and a CDR H3 with an amino acid sequenceof SEQ ID NO:
 9. 23. The isolated antibody of any one of claim 1 to 7,or 22, which has a V_(L) domain comprising a CDR L1 with an amino acidsequence of SEQ ID NO: 12, a CDR L2 with an amino acid sequence of SEQID NO: 14, and a CDR L3 with an amino acid sequence of SEQ ID NO: 16.24. The isolated antibody of any one of claims 1 to 7, which has a V_(H)domain comprising a CDR H1 with an amino acid sequence of SEQ ID NO: 20,a CDR H2 with an amino acid sequence of SEQ ID NO: 22, and a CDR H3 withan amino acid sequence of SEQ ID NO: 24, or a V_(H) comprising a CDR H1with an amino acid sequence of SEQ ID NO: 21 a CDR H2 with an amino acidsequence of SEQ ID NO: 23, and a CDR H3 with an amino acid sequence ofSEQ ID NO:
 25. 25. The isolated antibody of any one of claim 1 to 7, or23, which has a V_(L) domain comprising a CDR L1 with an amino acidsequence of SEQ ID NO: 28, a CDR L2 with an amino acid sequence of SEQID NO: 30, and a CDR L3 with an amino acid sequence of SEQ ID NO: 32.26. An isolated antibody of claims 1 to 7, comprising a V_(H) domainencoded by a nucleotide sequence that is at least 90% identical to thenucleotide sequence of SEQ ID NOs: 2 or 18, and/or a V_(L) domainencoded by a nucleotide sequence that is at least 90% identical to thenucleotide sequence of SEQ ID NOs: 19 or
 26. 27. The isolated antibodyof claim 26, wherein nucleotide sequences encoding the V_(H) and/or theV_(L) domains are at least 95% identical to the nucleotide sequences ofSEQ ID NOs: 2 or 18, or SEQ ID NOs: 10 or 26, respectively.
 28. Theisolated antibody of claim 27, wherein the nucleotide sequences encodingthe V_(H) and/or the V_(L) domains are at least 98% identical to SEQ IDNOs: 2 or 18, or SEQ ID NOs: 10 or 26, respectively.
 29. The isolatedantibody of any one of claims 22 to 28, which has human antibodyframework regions.
 30. The isolated antibody of any one of claims 22 to29, which has a human antibody constant domain.
 31. The isolatedantibody of any one of claims 1 to 30, wherein the antibody is an IgG,IgM, IgA antibody, or an antigen binding fragment thereof.
 32. Theisolated antibody of any one of claims 1 to 31, wherein the antibody isa Fab′, a F(ab′)2, a F(ab′)3, a monovalent scFv, a bivalent scFv, abiparatopic, or a single domain antibody.
 33. The isolated antibody ofany one of claims 1 to 32, which is a bispecific or biparatopicantibody.
 34. The isolated antibody of any one of claims 1 to 33,wherein the antibody is conjugated to an imaging agent, achemotherapeutic agent, a cytotoxic agent, an antineoplastic agent. or aradionuclide.
 35. An isolated nucleic acid molecule comprising anucleotide sequence of SEQ ID NOs: 2 or
 18. 36. An isolated nucleic acidmolecule comprising a nucleotide sequence of SEQ ID NO: 10 or
 26. 37. Anisolated nucleic acid molecule comprising a nucleotide sequence encodinga V_(H) domain and/or a V_(L) domain of an antibody of any one of claims1 to
 32. 38. A composition comprising the antibody of any one of claims1 to 34 in a pharmaceutically acceptable carrier, diluent, excipient, orvehicle.
 39. A method of treating a PD-L1 positive cancer in a subjectin need, comprising administering to a subject having a PD-L1 positivecancer an effective amount of the isolated antibody of any one of claims1-34.
 40. The method of claim 39, wherein the PD-L1 positive cancer is abreast cancer, lung cancer, head & neck cancer, prostate cancer,esophageal cancer, tracheal cancer, skin cancer brain cancer, livercancer, bladder cancer, stomach cancer, pancreatic cancer, ovariancancer, uterine cancer, cervical cancer, testicular cancer, coloncancer, rectal cancer or skin cancer.
 41. The method of claim 39,wherein the PD-L1 positive cancer is a hematological cancer.
 42. Themethod of any one of claims 39 to 41, which comprises administering incombination with said isolated antibody a second isolated antibody whichselectively binds to glycosylated PD-L1 relative to unglycosylatedPD-L1.
 43. The method of any one of claims 39 to 41, wherein theantibody is in a pharmaceutically acceptable composition.
 44. The methodof claim 43, wherein the antibody is administered systemically,intravenously, intradermally, intratumorally, intramuscularly,intraperitoneally, subcutaneously or locally.
 45. The method of claims39 to 44, further comprising administering in combination with saidisolated antibody at least a second anticancer therapy to the subject.46. The method of claim 45, wherein the second anticancer therapy is asurgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonaltherapy, immunotherapy or cytokine therapy.
 47. The isolated antibody ofclaim 34, wherein the antibody is conjugated to an antineoplastic agentto produce an antibody-drug conjugate (ADC).
 48. The isolated antibodyof claim 47, wherein the antineoplastic agent is an agent that inhibitstubulin polymerization.
 49. The isolated antibody of claim 48, whereinthe agent is a maytansinoid or an auristatin.
 50. The isolated antibodyof claim 49, wherein the maytansinoid is DM1(N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine) or DM4(N2′-deacetyl-n2′-(4-Mercapto-4-Methyl-1-oxopentyl)-6-MethylMaytansine.51. The isolated antibody of claim 49, wherein the auristatin ismonomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). 52.The isolated antibody of claim 51, wherein the auristatin is MMAE. 53.The isolated antibody of claim 52, wherein the antibody is chemicallyconjugated to a maleimide and caproic acid (MC) attachment group, whichis chemically conjugated to a cathepsin-cleavable linker, which ischemically conjugated to a paraminobenzoic acid (PAB) spacer, which ischemically conjugated to MMAE, thereby forming the ADC.
 54. The isolatedantibody of claim 53, wherein the cathepsin-cleavable linker isvaline-citruline (vc).
 55. The isolated antibody of claim 54, whereinthe antibody is STM073 or STM108.
 56. The isolated antibody of claim 55,wherein the antibody is STM108.
 57. An ADC comprising a dual functionanti-glycPD-L1 antibody chemically coupled to a cytotoxic drug.
 58. TheADC of claim 57, wherein the antibody is STM108 or STM073.
 59. The ADCof claim 57 or claim 58, wherein the cytotoxic drug is an agent thatinhibits tubulin polymerization.
 60. The ADC of claim 59, wherein theagent is a maytansinoid or an auristatin.
 61. The ADC of claim 60,wherein the maytansinoid is DM1 or DM4.
 62. The ADC of claim 60, whereinthe auristatin is MMAE or MMAF.
 63. The ADC of claim 62, wherein theauristatin is MMAE.
 64. The ADC of claim 63, wherein the antibody ischemically conjugated to an MC attachment group, which is chemicallyconjugated to a cathepsin-cleavable linker, which is chemicallyconjugated to a PAB spacer, which is chemically conjugated to MMAE,thereby forming the ADC.
 65. The ADC of claim 64, wherein thecathepsin-cleavable linker is valine-citruline (vc).
 66. The ADC ofclaim 65, wherein the antibody is STM108.
 67. An ADC comprisinganti-glycPD-L1 antibody STM108 chemically coupled to MMAE via acleavable linker.